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.

The multipass membrane protein APH-1, found in the gamma-secretase complex together with presenilin, nicastrin, and PEN-2, is essential for Notch signaling in Caenorhabditis elegans embryos and is required for intramembrane proteolysis of Notch and beta-amyloid precursor protein in mammalian and Drosophila cells. In C. elegans, a mutation of the conserved transmembrane Gly123 in APH-1 (mutant or28) leads to a notch/glp-1 loss-of-function phenotype. In this study, we show that the corresponding mutation in mammalian APH-1aL (G122D) disrupts the physical interaction of APH-1aL with hypoglycosylated immature nicastrin and the presenilin holoprotein as well as with mature nicastrin, presenilin, and PEN-2. The G122D mutation also reduced gamma-secretase activity in intramembrane proteolysis of membrane-tethered Notch. Moreover, we found that the conserved transmembrane Gly122, Gly126, and Gly130 in the fourth transmembrane region of mammalian APH-1aL are part of the membrane helix-helix interaction GXXXG motif and are essential for the stable association of APH-1aL with presenilin, nicastrin, and PEN-2. These findings suggest that APH-1 plays a GXXXG-dependent scaffolding role in both the initial assembly and subsequent maturation and maintenance of the active gamma-secretase complex.

The TAO (for thousand-and-one amino acids) protein kinases activate p38 mitogen-activated protein (MAP) kinase cascades in vitro and in cells by phosphorylating the MAP/ERK kinases (MEKs) 3 and 6. We found that TAO2 activity was increased by carbachol and that carbachol and the heterotrimeric G protein G␣ o could activate p38 in 293 cells. Using dominant interfering kinase mutants, we found that MEKs 3 and 6 and TAOs were required for p38 activation by carbachol or the constitutively active mutant G␣ o Q205L. To explore events downstream of TAOs, the effects of TAO2 on ternary complex factors (TCFs) were investigated. Transfection studies demonstrated that TAO2 stimulates phosphorylation of the TCF Elk1 on the major activating site, Ser 383 , and that TAO2 stimulates transactivation of Elk1 and the related TCF, Sap1. Reporter activity was reduced by the p38-selective inhibitor SB203580. Taken together, these studies suggest that TAO protein kinases relay signals from carbachol through heterotrimeric G proteins to the p38 MAP kinase, which then activates TCFs in the nucleus. TAO11 (thousand-and-one amino acids 1) and TAO2 are protein kinases that were originally identified based on their similarity to the yeast p21-activated protein kinase Ste20p, a protein kinase upstream in a mitogen-activated protein kinase (MAPK) pathway in yeast (1, 2). JIK is a third TAO-like kinase (3). The Ste20p family contains a diverse array of protein kinases, several of which have been shown to act upstream of MAPKs (4). TAO1 and TAO2 each contain over 1000 residues with catalytic domains at their N termini. TAOs activate MAPK pathways because they have MAP kinase kinase kinase (MAP3K) activity; they phosphorylate the p38-activating kinases called MAP/ERK kinases (MEKs, also known as MKKs or MAP2Ks) 3 and 6, which then phosphorylate p38 (1, 2, 5). The preferential phosphorylation of these two MEKs arises in part because TAOs dock to these MEKs through a region Cterminal to the TAO kinase domain (2, 5).Ternary complex factors (TCFs) are among the transcription factors under the control of MAPKs (6 -12). Upon phosphorylation by MAPKs, TCFs form a complex with the serum response factor at the serum response element, thereby stimulating the transcription of c-fos and other genes containing this element. The TCFs, Elk1, Sap1, and Sap2, contain a conserved C-terminal transactivation domain with multiple (S/T)P motifs, which are minimal consensus sites for MAPK phosphorylation. Elk1 can be phosphorylated by at least three MAPK subgroups, ERK1/2, c-Jun-N-terminal kinases/stress-activated protein kinases (JNK/SAPKs), and p38. In contrast, Sap1 can be phosphorylated by ERK1/2, ERK5, and p38, but not JNK/ SAPKs (7, 12, 13). We explored downstream actions of TAO2 by examining its effects on transactivation of TCFs. Cotransfection studies and reporter assays demonstrated that TAO2 enhances phosphorylation and transactivation of TCFs in 293 cells. TAO2-dependent transactivation of TCFs was inhibited by the p38 selective inhibitor SB203580, suggest...

Notch is a transmembrane receptor that controls a diverse array of cellular processes including cell proliferation, differentiation, survival, and migration. The cellular outcome of Notch signaling is dependent on extracellular and intracellular signals, but the complexities of its regulation are not well understood. Canonical Notch signaling involves ligand association that triggers sequential and regulated proteolysis of Notch at several sites. Ligand-dependent proteolysis at the S2 site removes the bulk of the extracellular domain of Notch. Subsequent ␥-secretase-mediated intramembrane proteolysis of the remaining membrane-tethered Notch fragment at the S3 site produces a nuclear-destined Notch intracellular domain (NICD). Here we show that following ␥-secretase cleavage, Notch is proteolyzed at a novel S5 site. We have identified this S5 site to be eight amino acids downstream of the S3 site. The Notch signaling pathway is evolutionarily conserved and plays a fundamental role in communication between adjacent cells to influence differentiation, proliferation, survival, and migration (1-3). During its maturation and function as a signal transducer, the Notch receptor undergoes proteolysis by three different enzymes. Notch is processed initially by a furin-like convertase in the trans-Golgi at the S1 site to generate an extracellular domain and a membrane-bound domain, which remain associated during their transport to the plasma membrane.Binding of a ligand to the extracellular domain of Notch stimulates proteolysis of Notch at the S2 site by the metalloprotease tumor necrosis factor ␣-converting enzyme (TACE) and produces a transmembrane fragment referred to as Notch extracellular truncation (N⌬E) 3 (4, 5). The membrane-tethered N⌬E is recruited to the ␥-secretase complex for S3 site cleavage to generate the Notch intracellular domain (NICD) (6 -8). The resultant NICD translocates into the nucleus, where it associates with the transcription factor CBF-1 and with coactivators, such as Mastermind, to initiate gene transcription (9 -12). Notch has mostly been characterized by its transcriptional role in the nucleus, but several studies suggest that it may have functional roles in the cytoplasm (13, 14) as well as in the mitochondria (15)(16)(17)(18)(19)(20) where, in the latter, Notch acts as an interpreter of cellular cues. In this study, we provide evidence that following cleavage by ␥-secretase, NICD is further proteolyzed at a novel S5 site. We identify the protease responsible for this cleavage as the mitochondrial intermediate peptidase (MIPEP), which generates ⌬NICD ( 1752 RQHGQ-…). We showed that Notch function can be modulated by MIPEP-mediated cleavage. Our findings raise the possibility that S5 site proteolysis represents a novel regulatory component of Notch signaling. EXPERIMENTAL PROCEDURES Materials-Chemicals used included ␥-secretase inhibitor N-[N-(3,5-Difluorophenylacetyl)-L-alanyl]-S-phenylglycine t-bu-tyl ester (DAPT) (Calbiochem/EMD) and the EDTA-free complete protease inhibitor mixture tabl...