The release of amyloid precursor protein (APP) intracellular domain (AICD) may be triggered by extracellular cues through γ-secretase-dependent cleavage. AICD binds to Fe65, which may have a role in AICD-dependent signalling; however, the functional ligand has not been characterized. In this study, we have identified TAG1 as a functional ligand of APP. We found that, through an extracellular interaction with APP, TAG1 increased AICD release and triggered Fe65-dependent activity in a γ-secretasedependent manner. TAG1, APP and Fe65 colocalized in the neural stem cell niche of the fetal ventricular zone. Neural precursor cells from TAG1 -/-, APP -/-and TAG1 -/-;APP -/-mice had aberrantly enhanced neurogenesis, which was significantly reversed in TAG1 -/-mice by TAG1 or AICD but not by AICD mutated at the Fe65 binding site. Notably, TAG1 reduced normal neurogenesis in Fe65 +/+ mice. Abnormally enhanced neurogenesis also occurred in Fe65 -/-mice but could not be reversed by TAG1. These results describe a TAG1-APP signalling pathway that negatively modulates neurogenesis through Fe65.The γ-secretase proteolytic complex cleaves a wide spectrum of type-1 transmembrane protein substrates, including Notch and APP, by regulated intramembrane proteolysis (RIP) to release their intracellular domains 1 . Ligand-binding to the substrate protein is one mechanism by which this cleavage is regulated. When a ligand binds to Notch, RIP stimulates the release of the intracellular domain of Notch (NICD), which interacts with the transcription factor CSL (CBF1, Suppressor of Hairless and Lag1; ref. 1). Similar transcriptional activity or regulation has been proposed for the intracellular domains cleaved from other γ-secretase substrates, including AICD, which is cleaved from APP 1 . It is therefore important to understand the physiological mechanisms regulating cleavage of AICD.Glycophosphatidylinositol (GPI)-linked proteins are anchored to the outer leaflet of the plasma membrane and mediate the dynamic remodelling of membranes during cell-cell interactions. In the central nervous system (CNS), GPI-linked recognition molecules, such as TAG1, NB-3 and F3, have been implicated in key developmental events, including selective axonal fasciculation, neural cell adhesion and migration, and neurite outgrowth 2 . Recently, we identified F3 and its homologue NB-3 as functional ligands for the Notch receptor and we showed that their interaction with each other is involved in oligodendrocyte differentiation through activation of the transcriptional factor Deltex1 (refs 3, 4). Given that RIP processing of APP is strikingly similar to that of the Notch receptor 5 , knowledge of the interaction between F3 and the Notch receptor has led us to ask whether members of the F3 family may act as APP ligands. RESULTS TAG1 and APP bind to each otherTo investigate the potential interaction between APP and members of the F3 subfamily, cell adhesion assays were performed. When F3-transfected CHO (CHOF3) cells or non-transfected CHO cells were seeded onto c...

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We investigated the molecular and cellular actions of receptor protein tyrosine phosphatase (PTP) α in integrin signaling using immortalized fibroblasts derived from wild-type and PTPα-deficient mouse embryos. Defects in PTPα−/− migration in a wound healing assay were associated with altered cell shape and focal adhesion kinase (FAK) phosphorylation. The reduced haptotaxis to fibronectin (FN) of PTPα−/− cells was increased by expression of active (but not inactive) PTPα. Integrin-mediated formation of src–FAK and fyn–FAK complexes was reduced or abolished in PTPα−/− cells on FN, concomitant with markedly reduced phosphorylation of FAK at Tyr397. Reintroduction of active (but not inactive) PTPα restored FAK Tyr-397 phosphorylation. FN-induced cytoskeletal rearrangement was retarded in PTPα−/− cells, with delayed filamentous actin stress fiber assembly and focal adhesion formation. This mimicked the effects of treating wild-type fibroblasts with the src family protein tyrosine kinase (Src-PTK) inhibitor PP2. These results, together with the reduced src/fyn tyrosine kinase activity in PTPα−/− fibroblasts (Ponniah et al., 1999; Su et al., 1999), suggest that PTPα functions in integrin signaling and cell migration as an Src-PTK activator. Our paper establishes that PTPα is required for early integrin-proximal events, acting upstream of FAK to affect the timely and efficient phosphorylation of FAK Tyr-397.

Glycosyl phosphatidylinositol (GPI)–linked receptors and receptor protein tyrosine phosphatases (RPTPs), both play key roles in nervous system development, although the molecular mechanisms are largely unknown. Despite lacking a transmembrane domain, GPI receptors can recruit intracellular src family tyrosine kinases to receptor complexes. Few ligands for the extracellular regions of RPTPs are known, relegating most to the status of orphan receptors. We demonstrate that PTPα, an RPTP that dephosphorylates and activates src family kinases, forms a novel membrane-spanning complex with the neuronal GPI-anchored receptor contactin. PTPα and contactin associate in a lateral (cis) complex mediated through the extracellular region of PTPα. This complex is stable to isolation from brain lysates or transfected cells through immunoprecipitation and to antibody-induced coclustering of PTPα and contactin within cells. This is the first demonstration of a receptor PTP in a cis configuration with another cell surface receptor, suggesting an additional mode for regulation of a PTP. The transmembrane and catalytic nature of PTPα indicate that it likely forms the transducing element of the complex, and we postulate that the role of contactin is to assemble a phosphorylation-competent system at the cell surface, conferring a dynamic signal transduction capability to the recognition element.

p53 mediates DNA damage-induced cell-cycle arrest, apoptosis, or senescence, and it is controlled by Mdm2, which mainly ubiquitinates p53 in the nucleus and promotes p53 nuclear export and degradation. By searching for the kinases responsible for Mdm2 S163 phosphorylation under genotoxic stress, we identified S6K1 as a multifaceted regulator of Mdm2. DNA damage activates mTOR-S6K1 through p38a MAPK. The activated S6K1 forms a tighter complex with Mdm2, inhibits Mdm2-mediated p53 ubiquitination, and promotes p53 induction, in addition to phosphorylating Mdm2 on S163. Deactivation of mTOR-S6K1 signalling leads to Mdm2 nuclear translocation, which is facilitated by S163 phosphorylation, a reduction in p53 induction, and an alteration in p53-dependent cell death. These findings thus establish mTOR-S6K1 as a novel regulator of p53 in DNA damage response and likely in tumorigenesis. S6K1-Mdm2 interaction presents a route for cells to incorporate the metabolic/energy cues into DNA damage response and links the aging-controlling Mdm2-p53 and mTOR-S6K pathways.