Bidirectional signaling triggered by interacting ephrinB receptors (EphB) and ephrinB ligands is crucial for development and function of the vascular and nervous systems. A signaling cascade triggered by this interaction involves activation of Src kinase and phosphorylation of ephrinB. The mechanism, however, by which EphB activates Src in the ephrinB-expressing cells is unknown. Here we show that EphB stimulates a metalloproteinase cleavage of ephrinB2, producing a carboxy-terminal fragment that is further processed by PS1/c-secretase to produce intracellular peptide ephrinB2/CTF2. This peptide binds Src and inhibits its association with inhibitory kinase Csk, allowing autophosphorylation of Src at residue tyr418. EphrinB2/CTF2-activated Src phosphorylates ephrinB2 and inhibits its processing by c-secretase. These data show that the PS1/c-secretase system controls Src activation and ephrinB phosphorylation by regulating production of Src activator ephrinB2/CTF2. Accordingly, csecretase inhibitors prevented the EphB-induced sprouting of endothelial cells and the recruitment of Grb4 to ephrinB. PS1 FAD and c-secretase dominant-negative mutants inhibited the EphB-induced cleavage of ephrinB2 and Src autophosphorylation, raising the possibility that FAD mutants interfere with the functions of Src and ephrinB2 in the CNS.
Binding of EphB receptors to ephrinB ligands on the surface of adjacent cells initiates signaling cascades that regulate angiogenesis, axonal guidance, and neuronal plasticity. These functions require processing of EphB receptors and removal of EphB-ephrinB complexes from the cell surface, but the mechanisms involved are poorly understood. Here we show that the ectodomain of EphB2 receptor is released to extracellular space following cleavage after EphB2 residue 543. The remaining membrane-associated fragment is cleaved by the presenilin-dependent ␥-secretase activity after EphB2 residue 569 releasing an intracellular peptide that contains the cytoplasmic domain of EphB2. This cleavage is inhibited by presenilin 1 familial Alzheimer disease mutations. Processing of EphB2 receptor depends on specific treatments: ephrinB ligand-induced processing requires endocytosis, and the ectodomain cleavage is sensitive to peptide inhibitor N-benzyloxycarbonyl-Val-Leu-leucinal but insensitive to metalloproteinase inhibitor GM6001. The ligand-induced processing takes place in endosomes and involves the rapid degradation of the extracellular EphB2. EphrinB ligand stimulates ubiquitination of EphB2 receptor. Calcium influx-and N-methyl-D-aspartic acid-induced processing of EphB2 is inhibited by GM6001 and ADAM10 inhibitors but not by N-benzyloxycarbonyl-Val-Leu-leucinal. This processing requires no endocytosis and promotes rapid shedding of extracellular EphB2, indicating that it takes place at the plasma membrane. Our data identify novel cleavages and modifications of EphB2 receptor and indicate that specific conditions determine the proteolytic systems and subcellular sites involved in the processing of this receptor.The Ephrin (Eph) 2 receptors are the largest family of receptor tyrosine kinase proteins. They bind membrane ligand proteins, called ephrins, on adjacent cells forming multimeric clusters that bridge juxtaposed cells. These binding interactions trigger signaling cascades in both the receptor-expressing cells (forward signaling) and the ligand-expressing cells (reverse signaling) stimulating functions that modulate cell morphogenesis, tissue patterning, and angiogenesis (1-4). In the developing central nervous system, binding of ephrin ligands to Eph receptors regulates axon guidance and synapse formation (5, 6). Paradoxically, depending on specific conditions such as the expression levels of Eph receptors and their ligands, signaling events initiated by the Eph-ephrin interactions can lead either to increased cell-cell adhesion or to repulsion and separation of the involved cells (4). In the adult brain, the Eph-ephrin systems regulate memory-related functions, including synaptic structure and long term potentiation (5,7,8). There are two subclasses of Eph receptors, EphA and EphB, which are selectively activated by ephrinA and ephrinB ligands, respectively, although exceptions to this rule have been observed (4).The EphB-ephrinB system regulates the development of many tissues, including the vasculature and the c...
The familial Alzheimer's disease gene product -amyloid (A) precursor protein (APP) is processed by the -and ␥-secretases to produce A as well as AID (APP Intracellular Domain) which is derived from the extreme carboxyl terminus of APP. AID was originally shown to lower the cellular threshold to apoptosis and more recently has been shown to modulate gene expression such that it represses Notch-dependent gene expression while in combination with Fe65 it enhances gene activation. Here we report that the two other members of the APP family, -amyloid precursor-like protein-1 and -2 (APLP1 and APLP2), are also processed by the ␥-secretase in a Presenilin 1-dependent manner. Furthermore, the extreme carboxyl-terminal fragments produced by this processing (here termed APP-like Intracellular Domain or ALID1 and ALID2) are able to enhance Fe65-dependent gene activation, similar to what has been reported for AID. Considering that only APP and not the APLPs have been linked to familial Alzheimer's disease (AD), this data should help in understanding the physiologic roles of the APP family members and in differentiating these functions from the pathologic role of APP in Alzheimer's disease.The APP 1 family consists of three family members, APP, APLP1, and APLP2 (1-4). Most research on the family has been focused on APP because it has been directly implicated in Alzheimer's disease (5, 6). APP undergoes extensive proteolytic processing along two major pathways. APP can be cleaved extracellularly by the ␣-secretase creating a C83 membranebound intermediate, which is subsequently cleaved by the ␥-secretase releasing a non-amyloidogenic fragment termed p3. Alternatively, APP can be cleaved extracellularly by the -secretase forming a C99 membrane-bound intermediate, which is subsequently cleaved by the ␥-secretase releasing the amyloidogenic A fragment. A is the major component of the amyloid plaques found in the brains of patients with AD and is considered to be the major underlying cause of the disease. An additional peptide termed AID, extending from the ␥-secretase cleavage site to the carboxyl terminus of APP, is also produced following ␥-secretase cleavage regardless of whether it was preceded by ␣ or  cleavage. This AID peptide was first identified in the brains of patients with AD and normal controls, and was shown to either induce or sensitize cells to apoptosis (7,8). More recently AID has been found to participate in transcription (9 -12) by activating gene expression in combination with Fe65 and repressing activation of genes induced by Notch. These data have opened the important possibility that just as Notch undergoes regulated intramembranous proteolysis (13) by the ␥-secretase, so does APP.Much less is known about APLP1 and APLP2, the two other APP family members. The human APLP1 gene is located on chromosome 19q13.1 (14), and APLP2 is located on chromosome 11q23-q25 (15). Although no clear neuronal functions have been found for these molecules, APLP1 has been implicated in synaptogenesis (16), and recomb...
The amyloid- protein precursor (APP) is a type I transmembrane molecule that undergoes several finely regulated cleavage events. The physiopathological relevance of APP derives from the fact that its aberrant processing strongly correlates with the onset of Alzheimer's disease (AD). AD is a neurodegenerative disorder characterized by neuronal cell death, loss of synapses, and deposition of misfolded protein plaques in the brain; the main constituent of these plaques is the amyloid- peptide, a 40 -42 amino-acid-long protein fragment derived by APP upon two sequential processing events. Mutations in the genes encoding for APP and some of the enzymes responsible for its processing are strongly associated with familial forms of early onset AD. Therefore, the elucidation of the mechanisms underlying APP metabolism appears crucial to understanding the basis for the onset of AD. Apart from A, upon processing of APP other fragments are generated. The long extracellular domain is released in the extracellular space, whereas the short cytoplasmic tail, named APP intracellular domain (AID) is released intracellularly. AID appears be involved in several cellular processes, apoptosis, calcium homeostasis, and transcriptional regulation. We have recently reported the cloning and characterization of different isoforms of AID associated protein-1 (AIDA-1), a novel AID-binding protein. Here we further analyzed the interaction between several AIDA-1 isoforms and the cytoplasmic tail of APP. Our data demonstrated that the interaction between the two molecules is regulated by alternative splicing of the AIDA-1 proteins. Furthermore, we provide data supporting a possible function for AIDA-1a as a modulator of APP processing.Alzheimer's disease is a neuropathological disorder characterized by dementia, memory loss, neuronal apoptosis, and, eventually, death of the affected individuals (1). Studies on familial forms of Alzheimer's disease revealed the crucial role played by mutations in genes encoding for APP 1 and presenilins in the pathogenesis of the disease (2-9). Presenilins are part of the ␥-secretasome, an enzymatic complex responsible for the intramembranous proteolysis of several transmembrane receptors, among which is APP (10 -20). Other enzymes, named -and ␣-secretases, cleave APP in its extracellular region releasing soluble N-terminal fragments (21, 22). The above-mentioned mutations in presenilins and APP are the genetic basis for familial forms of Alzheimer's disease, and they all result in aberrant processing of APP (10,(23)(24)(25). A lot of scientific interest has been more recently focused on studying potential functions for the intracellular domain of APP (AID), which is released upon cleavage by ␥-secretase. AID has been shown to be a pro-apoptotic molecule (26), to play a role in intracellular calcium homeostasis (27), to inhibit Notch signaling (28), and to be required for the activation and potential transcriptional activity of the adaptor proteins Fe65 (29,30) and Jun N-terminal kinase inter...
Alzheimer's disease (AD) is genetically linked to the processing of amyloid  protein precursor (APP). Aside from being the precursor of the amyloid  (A) found in plaques in the brains of patients with AD, little is known regarding the functional role of APP. We have recently reported biochemical evidence linking APP to the JNK signaling cascade by finding that JNK-interacting protein-1 (JIP-1) binds APP. In order to study the functional implications of this interaction we assayed the carboxyl-terminal of APP for phosphorylation. We found that the threonine 668 within the APP intracellular domain (AID or elsewhere AICD) is indeed phosphorylated by JNK1. We surprisingly found that although JIP-1 can facilitate this phosphorylation, it is not required for this process. We also found that JIP-1 only facilitated phosphorylation of APP but not of the two other family members APLP1 (amyloid precursorlike protein 1) and APLP2. Understanding the connection between APP phosphorylation and the JNK signaling pathway, which mediates cell response to stress may have important implications in understanding the pathogenesis of Alzheimer's disease.Alzheimer's disease (AD) 1 is the most common neurodegenerative disease constituting approximately two thirds of all cases of dementia (1). AD is genetically linked to a few molecules, one being APP. APP is a type I transmembrane protein, which undergoes processing by the secretases to produce various fragments. Following processing by the -and ␥-secretases, the A fragment (from the  to ␥ sites) and AID (from the ␥ site to the carboxyl-terminal) are produced (2, 3). Recently, another ␥-secretase-dependent cleavage has been described to occur at the "epsilon" site, which lies within AID (4 -7). This would cause shorter AID fragments of either 49 or 50 amino acids. The pathologic cascade, which leads to clinical manifestations of AD, has not been identified fully; however, the "Amyloid Hypothesis" has been used to explain certain aspects of AD pathology. According to this hypothesis, the accumulation of A is the primary event that leads to all subsequent events in the pathology of AD (8).However, considering that production of both A and AID are dependent on the ␥-secretase, we and others have attempted to understand the cellular effects of AID production. Indeed it has been found that AID is able to trigger apoptosis or lower the cell's threshold to other apoptotic stimuli (9). Furthermore, many proteins have been found to interact with AID such as X11, Fe65, mDab, Shc, Numb,. Use of "guilty by association" has been used to speculate possible roles for AID in the cell. One interacting protein, which has attracted much interest recently, is JNK-interacting protein (JIP)-1 (15). JIP-1 is a cytoplasmic protein that binds members of the JNK signaling cascade and scaffolds these proteins to allow for efficient activation of the JNK pathway (16). We and others (17)(18)(19) have shown that APP binds JIP-1.This biochemical connection between APP (and therefore AD), and JIP-...
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