Monoubiquitination and endocytosis direct gamma-secretase cleavage of activated Notch receptor

Activation of mammalian Notch receptor by its ligands induces TNFalpha-converting enzyme-dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent gamma-secretase activity. Here, we demonstrate that a new modification, a monoubiquitination, as well as clathrin-dependent endocytosis, is required for gamma-secretase processing of a constitutively active Notch derivative, DeltaE, which mimics the TNFalpha-converting enzyme-processing product. PS interacts with this modified form of DeltaE, DeltaEu. We identified the lysine residue targeted by the monoubiquitination event and confirmed its importance for activation of Notch receptor by its ligand, Delta-like 1. We propose a new model where monoubiquitination and endocytosis of Notch are a prerequisite for its PS-dependent cleavage, and discuss its relevance for other gamma-secretase substrates.     

Nicastrin functions as a gamma-secretase-substrate receptor

gamma-secretase catalyzes the intramembrane cleavage of amyloid precursor protein (APP) and Notch after their extracellular domains are shed by site-specific proteolysis. Nicastrin is an essential glycoprotein component of the gamma-secretase complex but has no known function. We now show that the ectodomain of nicastrin binds the new amino terminus that is generated upon proteolysis of the extracellular APP and Notch domains, thereby recruiting the APP and Notch substrates into the gamma-secretase complex. Chemical- or antibody-mediated blocking of the free amino terminus, addition of purified nicastrin ectodomain, or mutations in the ectodomain markedly reduce the binding and cleavage of substrate by gamma-secretase. These results indicate that nicastrin is a receptor for the amino-terminal stubs that are generated by ectodomain shedding of type I transmembrane proteins. Our data are consistent with a model where nicastrin presents these substrates to gamma-secretase and thereby facilitates their cleavage via intramembrane proteolysis.     

Spatial segregation of gamma-secretase and substrates in distinct membrane domains

Gamma-secretase facilitates the regulated intramembrane proteolysis of select type I membrane proteins that play diverse physiological roles in multiple cell types and tissue. In this study, we used biochemical approaches to examine the distribution of amyloid precursor protein (APP) and several additional gamma-secretase substrates in membrane microdomains. We report that APP C-terminal fragments (CTFs) and gamma-secretase reside in Lubrol WX detergent-insoluble membranes (DIM) of cultured cells and adult mouse brain. APP CTFs that accumulate in cells lacking gamma-secretase activity preferentially associate with DIM. Cholesterol depletion and magnetic immunoisolation studies indicate recruitment of APP CTFs into cholesterol- and sphingolipid-rich lipid rafts, and co-residence of APP CTFs, PS1, and syntaxin 6 in DIM patches derived from the trans-Golgi network. Photoaffinity cross-linking studies provided evidence for the preponderance of active gamma-secretase in lipid rafts of cultured cells and adult brain. Remarkably, unlike the case of APP, CTFs derived from Notch1, Jagged2, deleted in colorectal cancer (DCC), and N-cadherin remain largely detergent-soluble, indicative of their spatial segregation in non-raft domains. In embryonic brain, the majority of PS1 and nicastrin is present in Lubrol WX-soluble membranes, wherein the CTFs derived from APP, Notch1, DCC, and N-cadherin also reside. We suggest that gamma-secretase residence in non-raft membranes facilitates proteolysis of diverse substrates during embryonic development but that the translocation of gamma-secretase to lipid rafts in adults ensures processing of certain substrates, including APP CTFs, while limiting processing of other potential substrates.     

Regulated proteolytic processing of Tie1 modulates ligand responsiveness of the receptor-tyrosine kinase Tie2

Regulated ectodomain shedding followed by intramembrane proteolysis has recently been recognized as important in cell signaling and for degradation of several type I transmembrane proteins. The receptor-tyrosine kinase Tie1 is known to undergo ectodomain cleavage generating a membrane-tethered endodomain. Here we show Tie1 is a substrate for regulated intramembrane proteolysis. After Tie1 ectodomain cleavage the newly formed 45-kDa endodomain undergoes additional proteolytic processing mediated by gamma-secretase to generate an amino-terminal-truncated 42-kDa fragment that is subsequently degraded by proteasomal activity. This sequential processing occurs constitutively and is stimulated by phorbol ester and vascular endothelial growth factor. To assess the biological significance of regulated Tie1 processing, we analyzed its effects on angiopoietin signaling. Activation of ectodomain cleavage causes loss of phosphorylated Tie1 holoreceptor and generation of phosphorylated receptor fragments in the presence of cartilage oligomeric protein angiopoietin 1. A key function of gamma-secretase is in preventing accumulation of these phosphorylated fragments. We also find that regulated Tie1 processing modulates ligand responsiveness of the Tie-1-associated receptor Tie2. Activation of Tie1 ectodomain cleavage increases cartilage oligomeric protein angiopoietin 1 activation of Tie2. This correlates with increased ability of Tie2 to bind ligand after shedding of the Tie1 extracellular domain. A similar enhancement of ligand activation of Tie2 is seen when Tie1 expression is suppressed by RNA interference. Together these data indicate that Tie1, via its extracellular domain, limits the ability of ligand to bind and activate Tie2. Furthermore the data suggest that regulated processing of Tie1 may be an important mechanism for controlling signaling by Tie2.     

The insulin-like growth factor 1 (IGF-1) receptor is a substrate for gamma-secretase-mediated intramembrane proteolysis

Several type-1 membrane proteins undergo regulated intramembrane proteolysis resulting in the generation of biologically active protein fragments. Presenilin-dependant gamma-secretase activity is central to this event and includes amyloid precursor protein (APP), Notch and ErbB4 as substrates. Here we show that the insulin-like growth factor 1 receptor (IGF-IR) undergoes regulated intramembrane proteolysis. A metalloprotease-dependant ectodomain-shedding event generates a approximately 52 kDa IGF-IR-carboxyl terminal domain (CTD). The IGF-IR-CTD is consequentially a substrate for gamma-secretase cleavage, liberating a approximately 50 kDa intracellular domain (ICD) that can be inhibited by a specific gamma-secretase inhibitor. This study suggests that the IGF-IR is a substrate for gamma-secretase and may mediate a function independent of its role as a receptor tyrosine kinase.     

Proteomic profiling of gamma-secretase substrates and mapping of substrate requirements

The presenilin/gamma-secretase complex, an unusual intramembrane aspartyl protease, plays an essential role in cellular signaling and membrane protein turnover. Its ability to liberate numerous intracellular signaling proteins from the membrane and also mediate the secretion of amyloid-beta protein (Abeta) has made modulation of gamma-secretase activity a therapeutic goal for cancer and Alzheimer disease. Although the proteolysis of the prototypical substrates Notch and beta-amyloid precursor protein (APP) has been intensely studied, the full spectrum of substrates and the determinants that make a transmembrane protein a substrate remain unclear. Using an unbiased approach to substrate identification, we surveyed the proteome of a human cell line for targets of gamma-secretase and found a relatively small population of new substrates, all of which are type I transmembrane proteins but have diverse biological roles. By comparing these substrates to type I proteins not regulated by gamma-secretase, we determined that besides a short ectodomain, gamma-secretase requires permissive transmembrane and cytoplasmic domains to bind and cleave its substrates. In addition, we provide evidence for at least two mechanisms that can target a substrate for gamma cleavage: one in which a substrate with a short ectodomain is directly cleaved independent of sheddase association, and a second where a substrate requires ectodomain shedding to instruct subsequent gamma-secretase processing. These findings expand our understanding of the mechanisms of substrate selection as well as the diverse cellular processes to which gamma-secretase contributes.     

Regulated intramembrane proteolysis of the interleukin-1 receptor II by alpha-, beta-, and gamma-secretase

Ectodomain shedding and intramembrane proteolysis of the amyloid precursor protein (APP) by alpha-, beta- and gamma-secretase are involved in the pathogenesis of Alzheimer disease (AD). Increased proteolytic processing and secretion of another membrane protein, the interleukin-1 receptor II (IL-1R2), have also been linked to the pathogenesis of AD. IL-1R2 is a decoy receptor that may limit detrimental effects of IL-1 in the brain. At present, the proteolytic processing of IL-1R2 remains little understood. Here we show that IL-1R2 can be proteolytically processed in a manner similar to APP. IL-1R2 expressed in human embryonic kidney 293 cells first undergoes ectodomain shedding in an alpha-secretase-like manner, resulting in secretion of the IL-1R2 ectodomain and the generation of an IL-1R2 C-terminal fragment. This fragment undergoes further intramembrane proteolysis by gamma-secretase, leading to the generation of the soluble intracellular domain of IL-1R2. Intramembrane cleavage of IL-1R2 was abolished by a highly specific inhibitor of gamma-secretase and was absent in mouse embryonic fibroblasts deficient in gamma-secretase activity. Surprisingly, the beta-secretase BACE1 and its homolog BACE2 increased IL-1R2 secretion resulting in C-terminal fragments nearly identical to the ones generated by the alpha-secretase-like cleavage. This suggests that both proteases may act as alternative alpha-secretase-like proteases. Importantly, BACE1 and BACE2 did not cleave several other membrane proteins, demonstrating that both proteases do not contribute to general membrane protein turnover but only cleave specific proteins. This study reveals a similar proteolytic processing of IL-1R2 and APP and may provide an explanation for the increased IL-1R2 secretion observed in AD.     

The extracellular matrix protein MAGP-2 interacts with Jagged1 and induces its shedding from the cell surface

Elastic fibers are composed of the protein elastin and a network of 10-12-nm microfibrils, which are composed of several glycoproteins, including fibrillin-1, fibrillin-2, and MAGP1/2 (microfibril-associated glycoproteins-1 and -2). Although fibrillins and MAGPs covalently associate, we find that the DSL (Delta/Serrate/LAG2) protein Jagged1, an activating ligand for Notch receptor signaling, also interacts with MAGP-2 in both yeast two-hybrid and coimmunoprecipitation studies. Interaction between Jagged1 and MAGP-2 requires the epidermal growth factor-like repeats of Jagged1. MAGP-2 was found complexed with the Jagged1 extracellular domain shed from 293T cells and COS-7 cells coexpressing full-length Jagged1 and MAGP-2. MAGP-2 shedding of the Jagged1 extracellular domain was decreased by the metalloproteinase hydroxamate inhibitor BB3103 implicating proteolysis in its release. Although MAGP-2 also interacted with the other DSL ligands, Jagged2 and Delta1, they were not found associated with MAGP-2 in the conditioned media, identifying differential effects of MAGP-2 on DSL ligand shedding. The related microfibrillar protein MAGP-1 was also found to interact with DSL ligands but, unlike MAGP-2, was unable to facilitate the shedding of Jagged1. Our findings suggest that in addition to its role in microfibrils, MAGP-2 may also affect cellular differentiation through modulating the Notch signaling pathway either by binding to cell surface DSL ligands or by facilitating release and/or stabilization of a soluble extracellular form of Jagged1.     

Notch1 competes with the amyloid precursor protein for gamma-secretase and down-regulates presenilin-1 gene expression

Presenilin 1 (PS1) is a critical component of the gamma-secretase complex, which is involved in the cleavage of several substrates including the amyloid precursor protein (APP) and Notch1. Based on the fact that APP and Notch are processed by the same gamma-secretase, we postulated that APP and Notch compete for the enzyme activity. In this report, we examined the interactions between APP, Notch, and PS1 using the direct gamma-secretase substrates, Notch 1 Delta extracellular domain (N1DeltaEC) and APP carboxyl-terminal fragment of 99 amino acids, and measured the effects on amyloid-beta protein production and Notch signaling, respectively. Additionally, we tested the hypothesis that downstream effects on PS1 expression may coexist with the competition phenomenon. We observed significant competition between Notch and APP for gamma-secretase activity; transfection with either of two direct substrates of gamma-secretase led to a reduction in the gamma-cleaved products, Notch intracellular domain or amyloid-beta protein. In addition, however, we found that activation of the Notch signaling pathway, by either N1 Delta EC or Notch intracellular domain, induced down-regulation of PS1 gene expression. This finding suggests that Notch activation directly engages gamma-secretase and subsequently leads to diminished PS1 expression, suggesting a complex set of feedback interactions following Notch activation.     

Regulated intramembrane proteolysis of the p75 neurotrophin receptor modulates its association with the TrkA receptor

The generation of biologically active proteins by regulated intramembrane proteolysis is a highly conserved mechanism in cell signaling. Presenilin-dependent gamma-secretase activity is responsible for the intramembrane proteolysis of selected type I membrane proteins, including beta-amyloid precursor protein (APP) and Notch. A small fraction of intracellular domains derived from both APP and Notch translocates to and appears to function in the nucleus, suggesting a generic role for gamma-secretase cleavage in nuclear signaling. Here we show that the p75 neurotrophin receptor (p75NTR) undergoes presenilin-dependent intramembrane proteolysis to yield the soluble p75-intracellular domain. The p75NTR is a multifunctional type I membrane protein that promotes neurotrophin-induced neuronal survival and differentiation by forming a heteromeric co-receptor complex with the Trk receptors. Mass spectrometric analysis revealed that gamma-secretase-mediated cleavage of p75NTR occurs at a position located in the middle of the transmembrane (TM) domain, which is reminiscent of the amyloid beta-peptide 40 (Abeta40) cleavage of APP and is topologically distinct from the major TM cleavage site of Notch 1. Size exclusion chromatography and co-immunoprecipitation analyses revealed that TrkA forms a molecular complex together with either full-length p75 or membrane-tethered C-terminal fragments. The p75-ICD was not recruited into the TrkA-containing high molecular weight complex, indicating that gamma-secretase-mediated removal of the p75 TM domain may perturb the interaction with TrkA. Independent of the possible nuclear function, our studies suggest that gamma-secretase-mediated p75NTR proteolysis plays a role in the formation/disassembly of the p75-TrkA receptor complex by regulating the availability of the p75 TM domain that is required for this interaction.     

Interleukin-1 receptor type 1 is a substrate for gamma-secretase-dependent regulated intramembrane proteolysis

Biochemical and genetic studies have revealed that the presenilins interact with several proteins and are involved in the regulated intramembrane proteolysis of numerous type 1 membrane proteins, thereby linking presenilins to a range of cellular processes. In this study, we report the characterization of a highly conserved tumor necrosis factor receptor-associated factor-6 (TRAF6) consensus-binding site within the hydrophilic loop domain of presenilin-1 (PS-1). In coimmunoprecipitation studies we indicate that presenilin-1 interacts with TRAF6 and interleukin-1 receptor-associated kinase 2. Substitution of presenilin-1 residues Pro-374 and Glu-376 by site-directed mutagenesis greatly reduces the ability of PS1 to associate with TRAF6. By studying these interactions, we also demonstrate that the interleukin-1 receptor type 1 (IL-1R1) undergoes intramembrane proteolytic processing, mediated by presenilin-dependent gamma-secretase activity. A metalloprotease-dependent proteolytic event liberates soluble IL-1R1 ectodomain and produces an approximately 32-kDa C-terminal domain. This IL-1R1 C-terminal domain is a substrate for subsequent gamma-secretase cleavage, which generates an approximately 26-kDa intracellular domain. Specific pharmacological gamma-secretase inhibitors, expression of dominant negative presenilin-1, or presenilin deficiency independently inhibit generation of the IL-1R1 intracellular domain. Attenuation of gamma-secretase activity also impairs responsiveness to IL-1beta-stimulated activation of the MAPKs and cytokine secretion. Thus, TRAF6 and interleukin receptor-associated kinase 2 are novel binding partners for PS1, and IL-1R1 is a new substrate for presenilin-dependent gamma-secretase cleavage. These findings also suggest that regulated intramembrane proteolysis may be a control mechanism for IL-1R1-mediated signaling.     

Identification and characterization of presenilin-independent Notch signaling.

Presenilin (PS) proteins control the proteolytic cleavage that precedes nuclear access of the Notch intracellular domain. Here we observe that a partial activation of the HES1 promoter can be detected in PS1/PS2 (PS1/2) double null cells using Notch1 Delta E constructs or following Delta 1 stimulation, despite an apparent abolition of the production and nuclear accumulation of the Notch intracellular domain. PS1/2-independent Notch activation is sensitive to Numblike, a physiological inhibitor of Notch. PS1/2-independent Notch signaling is also inhibited by an active gamma-secretase inhibitor in the low micromolar range and is not inhibited by an inactive analogue, similar to PS-dependent Notch signaling. However, experiments using a Notch1-Gal4-VP16 fusion protein indicate that the PS1/2-independent activity does not release Gal4-VP16 and is therefore unlikely to proceed via an intramembranous cleavage. These data reveal that a novel PS1/2-independent mechanism plays a partial role in Notch signal transduction.     

Regulated Intramembrane Proteolysis of the Frontotemporal Lobar Degeneration Risk Factor,TMEM106B,by Signal Peptide Peptidase-like 2a (SPPL2a)

The sequential processing of single pass transmembrane proteins via ectodomain shedding followed by intramembrane proteolysis is involved in a wide variety of signaling processes, as well as maintenance of membrane protein homeostasis. Here we report that the recently identified frontotemporal lobar degeneration risk factor TMEM106B undergoes regulated intramembrane proteolysis. We demonstrate that TMEM106B is readily processed to an N-terminal fragment containing the transmembrane and intracellular domains, and this processing is dependent on the activities of lysosomal proteases. The N-terminal fragment is further processed into a small, rapidly degraded intracellular domain. The GxGD aspartyl proteases SPPL2a and, to a lesser extent, SPPL2b are responsible for this intramembrane cleavage event. Additionally, the TMEM106B paralog TMEM106A is also lysosomally localized; however, it is not a specific substrate of SPPL2a or SPPL2b. Our data add to the growing list of proteins that undergo intramembrane proteolysis and may shed light on the regulation of the frontotemporal lobar degeneration risk factor TMEM106B.     

A ligand-induced extracellular cleavage regulates gamma-secretase-like proteolytic activation of Notch1

Gamma-secretase-like proteolysis at site 3 (S3), within the transmembrane domain, releases the Notch intracellular domain (NICD) and activates CSL-mediated Notch signaling. S3 processing occurs only in response to ligand binding; however, the molecular basis of this regulation is unknown. Here we demonstrate that ligand binding facilitates cleavage at a novel site (S2), within the extracellular juxtamembrane region, which serves to release ectodomain repression of NICD production. Cleavage at S2 generates a transient intermediate peptide termed NEXT (Notch extracellular truncation). NEXT accumulates when NICD production is blocked by point mutations or gamma-secretase inhibitors or by loss of presenilin 1, and inhibition of NEXT eliminates NICD production. Our data demonstrate that S2 cleavage is a ligand-regulated step in the proteolytic cascade leading to Notch activation.