FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2072804535> ?p ?o ?g. }

• W2072804535 endingPage "2596" @default.
• W2072804535 startingPage "2586" @default.
• W2072804535 abstract "Article10 June 2004free access PS1 activates PI3K thus inhibiting GSK-3 activity and tau overphosphorylation: effects of FAD mutations Lia Baki Lia Baki Department of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Junichi Shioi Junichi Shioi Department of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Paul Wen Paul Wen Department of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Zhiping Shao Zhiping Shao Department of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Alexander Schwarzman Alexander Schwarzman Department of Psychiatry and Behavioural Sciences, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Miguel Gama-Sosa Miguel Gama-Sosa Department of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Rachael Neve Rachael Neve Departments of Psychiatry and Genetics, McLean Hospital, Harvard University, Belmont, MA, USA Search for more papers by this author Nikolaos K Robakis Corresponding Author Nikolaos K Robakis Department of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Lia Baki Lia Baki Department of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Junichi Shioi Junichi Shioi Department of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Paul Wen Paul Wen Department of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Zhiping Shao Zhiping Shao Department of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Alexander Schwarzman Alexander Schwarzman Department of Psychiatry and Behavioural Sciences, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Miguel Gama-Sosa Miguel Gama-Sosa Department of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Rachael Neve Rachael Neve Departments of Psychiatry and Genetics, McLean Hospital, Harvard University, Belmont, MA, USA Search for more papers by this author Nikolaos K Robakis Corresponding Author Nikolaos K Robakis Department of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Author Information Lia Baki1, Junichi Shioi1, Paul Wen1, Zhiping Shao1, Alexander Schwarzman2, Miguel Gama-Sosa1, Rachael Neve3 and Nikolaos K Robakis 1 1Department of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY, USA 2Department of Psychiatry and Behavioural Sciences, State University of New York at Stony Brook, Stony Brook, NY, USA 3Departments of Psychiatry and Genetics, McLean Hospital, Harvard University, Belmont, MA, USA *Corresponding author. Mount Sinai School of Medicine, NYU, One Gustave Levy Pl. Box 1229, Annenberg Bldg, Room 22-44A, New York, NY 10029, USA. Tel.: +1 212 241 9380; Fax: +1 212 831 1947; E-mail: [email protected] The EMBO Journal (2004)23:2586-2596https://doi.org/10.1038/sj.emboj.7600251 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Phosphatidylinositol 3-kinase (PI3K) promotes cell survival and communication by activating its downstream effector Akt kinase. Here we show that PS1, a protein involved in familial Alzheimer's disease (FAD), promotes cell survival by activating the PI3K/Akt cell survival signaling. This function of PS1 is unaffected by γ-secretase inhibitors. Pharmacological and genetic evidence indicates that PS1 acts upstream of Akt, at or before PI3K kinase. PS1 forms complexes with the p85 subunit of PI3K and promotes cadherin/PI3K association. Furthermore, conditions that inhibit this association prevent the PS1-induced PI3K/Akt activation, indicating that PS1 stimulates PI3K/Akt signaling by promoting cadherin/PI3K association. By activating PI3K/Akt signaling, PS1 promotes phosphorylation/inactivation of glycogen synthase kinase-3 (GSK-3), suppresses GSK-3-dependent phosphorylation of tau at residues overphosphorylated in AD and prevents apoptosis of confluent cells. PS1 FAD mutations inhibit the PS1-dependent PI3K/Akt activation, thus promoting GSK-3 activity and tau overphosphorylation at AD-related residues. Our data raise the possibility that PS1 may prevent development of AD pathology by activating the PI3K/Akt signaling pathway. In contrast, FAD mutations may promote AD pathology by inhibiting this pathway. Introduction Increased neuronal cell death, tau overphosphorylation and accumulation of neurofibrillary tangles (NFTs) and amyloid plaques are the main pathological hallmarks of Alzheimer's disease (AD) brains. The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays crucial roles in the transmission of survival signals in a wide range of cell types including neurons (for reviews, see Chan et al, 1999; Brunet et al, 2001). PI3K activates its downstream effector Akt/protein kinase B (Akt) by promoting its phosphorylation at residues serine 473 (Ser473) and threonine 308 (Thr308). Activated Akt, in turn, phosphorylates a wide range of substrates activating anti-apoptotic (survival) factors and inactivating pro-apoptotic factors (Brunet et al, 2001). The PI3K/Akt pathway is activated following recruitment of PI3K to the plasma membrane in response to a number of extracellular stimuli including growth factors (Brunet et al, 2001) and cadherin homophilic cell–cell adhesions, which result in the recruitment of PI3K to adhesion complexes (Pece et al, 1999; Kovacs et al, 2002; Tran et al, 2002; Yap and Kovacs, 2003). Akt downregulates the activities of glycogen synthase kinases 3α (GSK-3α) and 3β (GSK-3β) by phosphorylating the former at residue serine 21 (Ser21) and the latter at residue serine 9 (Ser9) (Cross et al, 1995; Kaytor and Orr, 2002). Increased GSK-3β activity has been implicated in neuronal cell death (Pap and Cooper, 1998; Hetman et al, 2000; Cross et al, 2001; Lucas et al, 2001) and tau overphosphorylation (Hanger et al, 1992; Hong et al, 1997; Pei et al, 1999; Lucas et al, 2001), while GSK-3α was recently implicated in the production of Aβ peptide, the principal protein component of amyloid plaques (Phiel et al, 2003). Presenilin1 (PS1), a multipass transmembrane protein important in development (Shen et al, 1997), is tightly linked to many cases of early-onset familial Alzheimer's disease (FAD) indicating a causal relationship between these mutants and AD pathology. As a result, the biological function of PS1 and the mechanism(s) involved in the pathogenic activities of PS1 mutants are under intense investigation. PS1 is cleaved in vivo to yield an N-terminal (PS1/NTF) fragment and a C-terminal (PS1/CTF) fragment that associate to form a functional heterodimer (Thinakaran et al, 1996). PS1 forms complexes with and promotes the γ-secretase-like processing of APP and several other type I transmembrane proteins (for review, see Fortini, 2002). Recently, we reported that PS1 binds cadherins and promotes cadherin–cadherin cell–cell adhesion interactions (Georgakopoulos et al, 1999; Baki et al, 2001). Ca2+ influx however, including activation of NMDA receptor, stimulates a PS1/γ-secretase-dependent processing of cadherins at the ε-cleavage site resulting in the production of gene expression signals (Marambaud et al, 2002, 2003). PS1 is expressed in neurons and plays crucial roles in the development of the CNS (Elder et al, 1996; Shen et al, 1997). In vitro experiments showed that overexpression of PS1 FAD mutants promotes apoptosis (Weihl et al, 1999; Leroy et al, 2003) and may decrease Akt activity (Weihl et al, 1999; Vestling et al, 2001). However, it remains unclear how PS1 promotes development (Shen et al, 1997) and cell survival (Hong et al, 1999) and whether these functions of PS1 are related to the mechanisms by which PS1 FAD mutants promote tau overphosphorylation and neuronal cell death (Irving and Miller, 1997; Takashima et al, 1998; Pigino et al, 2001). Here we show that PS1 promotes survival of confluent cell cultures by increasing cadherin/PI3K association, thus stimulating the PI3K/Akt cell survival signaling. This function of PS1 is independent of its γ-secretase activity. We also show that by promoting the PI3K/Akt signaling, PS1 downregulates GSK-3 activity and reduces overphosphorylation of tau at AD-related residues. In contrast, PS1 FAD mutations inhibit this signaling, thus compromising the cell survival function of PS1 and its ability to suppress GSK-3 and tau overphosphorylation. Results Absence of PS1 triggers density-dependent apoptosis Postconfluent E-cadherin-expressing PS1−/− cell cultures displayed increased cell death compared to control (PS1+/+) cells (Figure 1A). DNA fragmentation assays showed a clear laddering pattern in DNA from postconfluent PS1−/− cell cultures but not in DNA from postconfluent PS1+/+ cultures, indicating apoptotic cell death in the former cultures (Figure 1B). Accordingly, prominent TUNEL staining and progressive accumulation of pro-apoptotic cleaved (activated), caspase-3 were observed in postconfluent PS1−/− but not in PS1+/+ cells (Figures 1C and D, respectively). To exclude clonal effects, we established additional E-cadherin-transfected embryonic fibroblast clones derived from independent matings. Prominent TUNEL staining was observed in all postconfluent PS1−/− clones but not in PS1+/+ clones (data not shown). Apoptotic changes, revealed by the progressive activation of apoptotic caspase-3, are also observed in non-E-cadherin-transfected postconfluent PS1−/− cells (Figure 1E, lanes 1–6); E-cadherin, however, accelerates these changes as indicated by the presence of activated caspase-3 in 2 days postconfluent E-cadherin-transfected but not in nontransfected PS1−/− cell cultures (Figure 1E, compare lane 2 to lane 8). Thus, in the absence of PS1, cells undergo apoptosis following confluence. Since E-cadherin expression accelerates apoptosis, all following cell culture experiments were performed using E-cadherin-expressing cells. Figure 1.Absence of PS1 triggers density-dependent apoptosis. (A) Survival analysis of E-cadherin-transfected PS1+/+ or PS1−/− cells (continuous and dotted lines, respectively) cultured up to 4 days postconfluence. Mean values, ±s.e.m., from two independent experiments (each in triplicate) are presented. (B) Kinetics of the appearance of apoptotic (fragmented) DNA in nuclei of E-cadherin-transfected PS1+/+ and PS1−/− cells, cultured for up to 3 days postconfluence (Days PC). (C) Kinetics of the appearance of TUNEL-positive (apoptotic) nuclei in E-cadherin-transfected PS1+/+ and PS1−/− cells, cultured for up to 3 days postconfluence. (D) Lysates from 0-, 1- and 2-day postconfluent E-cadherin-transfected PS1+/+ and PS1−/− cells were analyzed with anti-N-cadherin (loading control) and anti-activated (cleaved) caspase-3 antibodies. N-cad: N-cadherin; casp3: activated caspase-3. (E) Lysates from 2- to 4-day postconfluent nontransfected PS1+/+ and PS1−/− cells (lanes 1–6) and from 2-day postconfluent E-cadherin-transfected PS1+/+ and PS1−/− cells (lanes 7 and 8) were analyzed with anti-b-tubulin (control) and anti-activated (cleaved) caspase-3 antibodies. Download figure Download PowerPoint PS1 inhibits apoptosis by activating PI3K/Akt signaling Inadequate activation of the PI3K/Akt signaling pathway may result in impaired transmission of survival signals leading to apoptosis (Brunet et al, 2001). PI3K kinase promotes cell survival by activating its downstream effector Akt, an event indicated by phosphorylation of Akt residue Ser473 (for review, see Chan and Tsichlis, 2001). Figure 2A shows that Akt Ser473 is underphosphorylated in PS1−/− cells suggesting that PS1 is needed for full activation of the PI3K/Akt cell survival pathway. Indeed, expression of PS1 in PS1−/− cells stimulated phosphorylation of Akt at Ser473 (Figure 2B, lanes 1 and 2). Pharmacological inhibition of PI3K activity strongly suppressed phosphorylation of Akt in PS1+/+ cells (Figure 2A, lane 3) and prevented the PS1-induced Akt phosphorylation in PS1−/− cells (Figure 2B, lane 3), suggesting that PI3K mediates the PS1-induced Akt phosphorylation. We next asked whether PS1-induced activation of the PI3K/Akt survival pathway is sufficient to rescue PS1−/− cells from apoptosis. Indeed, re-introduction of PS1 into PS1−/− cells decreased the number of annexin-positive (apoptotic) PS1−/− cells from 68 to 22% (Figure 2C) and strongly suppressed activation of apoptotic caspase-3 (Figure 2B, panel c, lanes 1 and 2). Pharmacological inhibition of PI3K activity prevented the PS1-induced inhibition of caspase-3 cleavage (Figure 2B, panel c) indicating that the anti-apoptotic function of PS1 depends on its ability to activate the PI3k/Akt signaling cascade. Figure 2.PS1 inhibits apoptosis by activating PI3K/Akt signaling. (A) Lysates from confluent E-cadherin-transfected PS1+/+ and PS1−/− fibroblasts, cultured in the absence or presence of the PI3K inhibitor LY24002, were analyzed for phosphorylated Akt at Ser473 (ph Akt) or total Akt. (B) PS1−/− cells infected with HSV vector (V) or HSV PS1 (PS1) were cultured in the presence or absence of PI3K inhibitor (LY24002). Lysates were analyzed as shown. ph Akt: phosphorylated Akt at Ser473; casp3: activated caspase-3; PS1-FL: full-length PS1; PS1-NTF: NTF fragment of PS1. (C) PS1−/− cells, transiently transfected either with EGFP vector (V) or with EGFP-PS1 (PS1), were subjected to annexin/7AAD staining and analyzed by flow cytometry. Only the EGFP-positive (transfected) cells are shown. Percentages refer to total EGFP-positive cells. Nonapoptotic cells are shown in the lower left square. A total of 10 000 EGFP-positive cells were analyzed. (D) PS1−/− cells were transfected with mutant (active) PI3K (PI3K*) or empty vector and lysates were analyzed as shown. Download figure Download PowerPoint The fact that inhibition of PI3K activity prevents the PS1-dependent Akt phosphorylation suggests that PS1 does not act directly on Akt but rather at an upstream step of the PI3K/Akt signaling pathway. PTEN phosphates negatively regulates the PI3K pathway by blocking signal transmission from PI3K to Akt. Examination of PTEN phosphorylation (Stambolic et al, 1998), however, showed that PTEN activity is not involved in the reduced Akt phosphorylation of PS1−/− cells (data not shown). Furthermore, a PI3K mutant with high constitutive activity (Rodriguez-Viciana et al, 1996) is able to stimulate Akt phosphorylation and to suppress activation of caspase-3 even in the absence of PS1 (Figure 2D). Together, these data indicate that PS1 promotes activation of the PI3K/Akt survival pathway by affecting a step at or before PI3K. PS1 regulates GSK-3 activity via the PI3K/Akt pathway To further explore the downstream physiological consequences of the PS1-dependent PI3K/Akt activation, we examined the phosphorylation of GSK-3α and GSK-3β kinases at the Akt-specific epitopes Ser21 and Ser9, respectively. Figure 3A shows that compared to wild-type (WT) cells, phosphorylation of both GSK-3 kinases is decreased in PS1−/− cells, suggesting that PS1 promotes the Akt-dependent phosphorylation/inactivation of both kinases (Cross et al, 1995; Kaytor and Orr, 2002). Indeed, re-introduction of PS1 in these cells increased phosphorylation of both kinases (Figure 3B, lanes 1 and 2) and this increase was prevented by pharmacological inhibition of either PI3K or Akt activities (Figure 3B, lanes 3 and 4). As expected, PS1 suppressed apoptotic caspace-3 and this suppression was abolished in the presence of either PI3K or Akt inhibitors (panel e). Together, these results show that PS1 increases GSK-3 phosphorylation via the PI3K/Akt signaling. Since this phosphorylation suppresses GSK-3 activity (Cross et al, 1995; Kaytor and Orr, 2002), our data indicate that PS1 downregulates the activity of both GSK-3 isoforms by stimulating the PI3K/Akt pathway. Figure 3.PS1 regulates GSK-3 activity via the PI3K/Akt pathway. (A) Lysates from confluent PS1+/+ or PS1−/− fibroblasts were analyzed for phosphorylation of GSK-3α (ph GSK-3α) and GSK-3β (ph GSK-3β) at Ser21 and Ser9, respectively. (B) PS1−/− cells infected with HSV vector (V) or HSV PS1 (PS1) were cultured in the presence or absence of either Akt inhibitor (Akt Inh) or PI3K inhibitor (LY24002). Lysates were analyzed as shown. PS1-NTF: NTF fragment of PS1. Download figure Download PowerPoint PS1-mediated activation of the PI3K–Akt pathway is independent of γ-secretase activity PS1 is important for the γ-secretase cleavage of many proteins including E- and N-cadherin. Inhibition of this cleavage by γ-secretase inhibitors or absence of PS1 results in the accumulation of cadherin fragments N-Cad/CTF1 and E-Cad/CTF1, which are substrates for the PS1/γ-secretase system (Marambaud et al, 2002, 2003). To explore any potential role of this system in the activation of PI3K/Akt signaling, we used two distinct γ-secretase inhibitors, L-685,458 and Compound E (see Materials and methods), to ask whether inhibition of γ-secretase affects Akt phosphorylation and apoptosis. Figure 4A shows that, in contrast to PS1−/− cells, PS1+/+ cells contain no detectable levels of the γ-secretase substrate N-Cad/CTF1 (lanes 1 and 2). As expected, treatment of PS1+/+ cells with either inhibitor resulted in the accumulation of N-Cad/CTF1 indicating inhibition of γ-secretase activity (Marambaud et al, 2003). These treatments, however, did not affect phosphorylation of Akt nor did they promote caspase-3 activation (Figure 4A, lanes 2–5). Figure 4.PS1-mediated activation of the PI3K–Akt pathway is independent of γ-secretase activity. (A) Confluent PS1−/− (lane 1) or PS1+/+ cells (lanes 2–5) were cultured overnight in the presence or absence of γ-secretase inhibitor XVIII (Compound E, lane 4), γ-secretase inhibitor L-685,458 (lane 5) or vehicle (DMSO, lane 3). Lysates were analyzed as shown. N-cad-FL: full-length N-cadherin; N-cad-CTF1: CTF1 fragment of N-cadherin (Marambaud et al, 2003). (B) PS1−/− cells were infected with HSV vector (V) or HSV PS1 (PS1) and cultured in the presence or absence of γ-secretase inhibitor. Lysates were analyzed as shown. E-cad-FL: full-length E-cadherin; E-cad-CTF1: CTF1 fragment of E-cadherin (Marambaud et al, 2002); PS1-CTF: CTF fragment of PS1. Download figure Download PowerPoint In a different approach we asked whether inhibition of γ-secretase activity prevents the PS1-induced activation of the PI3K pathway. Figure 4B (lanes 1 and 2) shows that re-introduction of PS1 in PS1−/− cells reduced E-cadherin fragment CTF1, increased Akt activation and reduced apoptotic caspase-3. However, although inhibitor L-685,458 almost completely prevented the γ-secretase activity-dependent metabolism of E-Cad/CTF1 (Marambaud et al, 2002), it affected neither the PS1-induced Akt phosphorylation nor the PS1-dependent inhibition of caspase-3 (Figure 4B, lanes 3 and 4). Together, the above data show that the PS1-dependent activation of the PI3K/Akt cell survival pathway is a novel function of PS1, unrelated to PS1-mediated γ-secretase activity. PS1 activates PI3K/Akt signaling by promoting association of the p85 subunit of PI3K with E- and N-cadherin The increased apoptosis manifested by confluent E-cadherin-transfected PS1−/− cells suggested that cadherins, proteins that associate and functionally interact with both PS1 and PI3K (Pece et al, 1999; Baki et al, 2001; Laprise et al, 2002; Tran et al, 2002), might be involved. Furthermore, PS1 promotes cadherin–cadherin adhesive interactions (Baki et al, 2001), a key prerequisite for cadherin-mediated activation of PI3K (Pece et al, 1999; Kovacs et al, 2002; Tran et al, 2002). To explore the possibility that cadherins are the mediators of the PS1 effect on PI3K/Akt signaling, we asked whether inhibition of adhesion function of either E- or N-cadherin would prevent the PS1-induced phosphorylation of Akt. Function-blocking antibodies against E- or N-cadherin inhibit cadherin–cadherin homophilic interactions and prevent cadherin-mediated activation of PI3K (Pece et al, 1999; Laprise et al, 2002; Tran et al, 2002). Figure 5A shows that either one of these antibodies suppressed phosphorylation of both Akt and its substrate GSK-3β in WT cells, but had no significant effect on the already low levels of phosphorylated Akt or GSK-3β of PS1−/− cells. Furthermore, a combination of the above antibodies prevented the PS1-induced phosphorylation of Akt (Figure 5B), showing that functional cadherins mediate the PS1-induced stimulation of PI3K/Akt signaling. Figure 5.PS1 activates the PI3K/Akt pathway by promoting association of the p85 subunit of PI3K with E- and N-cadherin. (A) Postconfluent PS1+/+ or PS1−/− cells were cultured in the presence of anti-N-cadherin and anti-E-cadherin antibodies (a-N and a-E, respectively) or isotypic IgG (IgG). Lysates were analyzed as shown. NT: no treatment. (B) PS1−/− cells were infected with HSV vector (V) or HSV PS1 (PS1) and cultured in the presence of both anti-E- and anti-N-cadherin antibodies (anti-E+anti-N) or isotypic IgG (IgG). Lysates were analyzed as shown. (C) Lysates from postconfluent PS1+/+ or PS1−/− cells were immunoprecipitated (IPed) with anti-PS1 (IP: PS1) or anti-p85/PI3K (IP: p85/PI3K) antibodies and obtained immunoprecipitates (IPs) were analyzed with anti-p85/PI3K or anti-PS1-CTF antibodies as shown. Asterisk denotes IgG. (D) Lysates from postconfluent PS1+/+ or PS1−/− cells were IPed with anti-E-cadherin or anti-p85/PI3K antibodies and obtained IPs were analyzed for E-cadherin or p85/PI3K. (E) Lysates as in (D) were IPed with anti-N-cadherin or anti-p85/PI3K antibodies and obtained IPs were analyzed as shown. (F) PS1−/− cells were infected with HSV vector (V) or HSV PS1 (PS1). Lysates were IPed with anti-E- or anti-N-cadherin antibodies and analyzed as shown. Asterisk denotes IgG. (G) Confluent PS1+/+ cells were preincubated for 45 min in the presence of 4 mM EGTA to break cadherin/PI3K complexes (Pece et al, 1999; Tran et al, 2002) and then cultures were switched to Ca2+-containing medium at time zero and followed for the times shown (lanes 1–3). Lysates were IPed with anti-E- or anti-N-cadherin antibodies and IPs were analyzed with anti-p85/PI3K, or anti-E- and anti-N-cadherin antibodies as shown. Steady-state (SS) levels of cadherin/PI3K complexes in the absence of EGTA were monitored in PS1+/+ and PS1−/− cultures (lanes 4 and 5, respectively). All cultures were in serum-free media. Download figure Download PowerPoint Cadherin-mediated activation of PI3K involves association of the p85 regulatory subunit of PI3K with either E- or N-cadherin (Pece et al, 1999; Baki et al, 2001; Laprise et al, 2002; Tran et al, 2002). Co-immunoprecipitation experiments reveal that PS1 forms a complex with the p85 subunit of PI3K (Figure 5C), raising the possibility that PS1 plays a role in the association of cadherins and p85. Indeed, association of p85 subunit of PI3K with either E- or N-cadherin is severely impaired in PS1−/− cells (Figures 5D and E, respectively). Moreover, re-introduction of PS1 into PS1−/− cells stimulates association of PI3K with both cadherins (Figure 5F). Thus, PS1 promotes association of the p85 subunit of PI3K with E- and N-cadherin. To further examine the involvement of the cadherin/PI3K complexes in the PS1-stimulated activation of the PI3K/Akt pathway, we asked whether disruption of these complexes suppresses Akt phosphorylation even in the presence of PS1 and whether de novo formation of the complexes would re-activate Akt. To this aim, we used a calcium switch approach to disrupt and re-form cadherin/PI3K complexes (Pece et al, 1999; Tran et al, 2002). Figure 5G shows that calcium deprivation of PS1+/+ cultures dissociates PI3K from both cadherins and strongly suppresses phosphorylation of Akt (compare lanes 1 and 4). Restoration of calcium results in a progressive re-formation of cadherin/PI3K complexes and restores Akt phosphorylation to steady-state levels (Figure 5G, lanes 1–4). In contrast, in the absence of PS1, both cadherin/PI3K complexes and Akt phosphorylation are significantly decreased despite the continuous presence of calcium (Figure 5G, compare lanes 4 and 5). Together, the above data show that PS1 promotes association of the p85 regulatory subunit of PI3K with E- and N-cadherin and that this association is necessary for the PS1-induced activation of the PI3K/Akt pathway. PS1 knockout embryos show reduced cadherin–PI3K complexes, impaired Akt activity and increased GSK-3-dependent phosphorylation of tau The physiological significance of PS1 for the cadherin–PI3K association and PI3K/Akt activation was examined in vivo using PS1 null mice. Figure 6A shows that, compared to WT embryos, PS1−/− embryos contain significantly lower amounts of the p85/E-cadherin complexes, whereas an even more dramatic reduction is observed in the levels of the N-cadherin/p85 complexes. Phosphorylation of both Akt and its substrate GSK-3β is also reduced in PS1−/− embryonic brains compared to WT littermates, indicating reduced activation of the PI3K/Akt pathway and increased GSK-3 activity in the absence of PS1 (Figure 6B). Figure 6.PS1 knockout embryos show reduced cadherin/PI3K complexes, decreased phosphorylation of Akt and GSK-3 and increased GSK-3-dependent phosphorylation of tau. (A) Total embryo homogenates prepared from PS1+/+ or PS1−/− mouse embryo littermates were immunoprecipitated with anti-E-cadherin (IP: E-cad) or anti-N-cadherin (IP: N-cad) antibodies and analyzed as shown. (B) Lysates were prepared from PS1+/+ or PS1−/− embryonic brains and analyzed for phosphorylated Akt and GSK-3β as shown. (C) Lysates were prepared from PS1+/− and PS1−/− mouse embryonic brains. The heat-stable fraction of lysates was analyzed with phosphorylation-dependent (PHF1, CP13) and phosphorylation-independent (TG5) anti-tau antibodies. Duplicate samples each from a littermate embryo are shown. Download figure Download PowerPoint GSK-3β (also called tau kinase 1) phosphorylates tau at several serine and threonine residues found hyperphosphorylated in AD brains (Hanger et al, 1992; Pei et al, 1999). The reduced phosphorylation of GSK-3β at Akt-dependent residues observed in brains from PS1−/− mice suggested an increased GSK-3 activity and prompted us to examine phosphorylation of tau at residues targeted by GSK-3β. Figure 6C shows that, consistent with the low levels of GSK-3β phosphorylation, brain tau of PS1 null mice is hyperphosphorylated at GSK-3-dependent epitopes compared to tau of WT (PS1+/+) brains. Thus, the absence of PS1 results in reduced cadherin/PI3K association, impaired PI3K/Akt signaling and in increased GSK-3 activity and tau phosphorylation in vivo. PS1 controls tau phosphorylation via the PI3K/Akt/GSK-3β pathway To further explore the role of PS1 in GSK-3β-dependent phosphorylation of tau, we transfected PS1+/+ and PS1−/− fibroblasts with the longest human tau isoform and then examined phosphorylation of tau residues Ser396/404 and Ser202 that are targets of GSK-3β and are overphosphorylated in AD brains (Sperber et al, 1995). Figure 7A shows that, in agreement with the low levels of phosphorylated GSK-3β in PS1−/− cells (Figure 7A, lanes 1 and 4, panels f and g), exogenous tau is hyperphosphorylated in these cells (Figure 7A, lanes 1 and 4, panels a–c) suggesting that absence of PS1 activity results in tau hyperphosphorylation. Since cells were transfected with one tau isoform, the multiple bands detected by anti-phosphorylated tau antibodies indicate multiple phosphorylation sites. LiCl, an inhibitor of GSK-3 activity, reduced tau phosphorylation in PS1−/− cells (Figure 7A, lanes 4 and 6, panels a–c), indicating involvement of GSK-3β activity in the observed tau overphosphorylation. Thus, absence of PS1 activity results in increased GSK-3-dependent tau phosphorylation. Inhibition of PI3K activity by LY24002 in PS1+/+ cells reduced GSK-3β phosphorylation and increased tau phosphorylation (Figure 7A, lanes 1 and 2, panels f–g and a–c, respectively), indicating that in the presence of PS1 maintenance of low levels of tau phosphorylation requires PI3K activity. In contrast, in the absence of PS1, LY24002 had no detectable effect on the already high levels of tau phosphorylation (Figure 7A, lanes 4 and 5, panels a–c). Figure 7.PS1 controls tau phosphorylation via the PI3K/Akt/GSK-3β pathway. (A) PS1+/+ or PS1−/− fibroblasts transiently expressing the longest human tau isoform were cultured in the presence or absence of PI3K inhibitor LY24002 (LY) or GSK-3 inhibitor LiCl. Lysates were analyzed for phosphorylation of tau (panels a–c), phosphoryla" @default.
• W2072804535 created "2016-06-24" @default.
• W2072804535 creator A5001425284 @default.
• W2072804535 creator A5028711885 @default.
• W2072804535 creator A5036668859 @default.
• W2072804535 creator A5051455921 @default.
• W2072804535 creator A5056187285 @default.
• W2072804535 creator A5060018103 @default.
• W2072804535 creator A5068723335 @default.
• W2072804535 creator A5084260138 @default.
• W2072804535 date "2004-06-10" @default.
• W2072804535 modified "2023-10-13" @default.
• W2072804535 title "PS1 activates PI3K thus inhibiting GSK-3 activity and tau overphosphorylation: effects of FAD mutations" @default.
• W2072804535 cites W1485366536 @default.
• W2072804535 cites W1526586150 @default.
• W2072804535 cites W1567624342 @default.
• W2072804535 cites W1785214340 @default.
• W2072804535 cites W1799837960 @default.
• W2072804535 cites W1863238068 @default.
• W2072804535 cites W1890393292 @default.
• W2072804535 cites W1964792461 @default.
• W2072804535 cites W1967550038 @default.
• W2072804535 cites W1986402843 @default.
• W2072804535 cites W1995434077 @default.
• W2072804535 cites W1998038367 @default.
• W2072804535 cites W1999910107 @default.
• W2072804535 cites W2004280410 @default.
• W2072804535 cites W2010710842 @default.
• W2072804535 cites W2012868552 @default.
• W2072804535 cites W2017680535 @default.
• W2072804535 cites W2031864556 @default.
• W2072804535 cites W2035120902 @default.
• W2072804535 cites W2045604110 @default.
• W2072804535 cites W2045834561 @default.
• W2072804535 cites W2049793641 @default.
• W2072804535 cites W2053265248 @default.
• W2072804535 cites W2053791137 @default.
• W2072804535 cites W2058138221 @default.
• W2072804535 cites W2059796847 @default.
• W2072804535 cites W2073128231 @default.
• W2072804535 cites W2073292164 @default.
• W2072804535 cites W2075175573 @default.
• W2072804535 cites W2077577830 @default.
• W2072804535 cites W2078680897 @default.
• W2072804535 cites W2082952854 @default.
• W2072804535 cites W2101741807 @default.
• W2072804535 cites W2103002807 @default.
• W2072804535 cites W2114358785 @default.
• W2072804535 cites W2122465857 @default.
• W2072804535 cites W2127113850 @default.
• W2072804535 cites W2127601584 @default.
• W2072804535 cites W2133485704 @default.
• W2072804535 cites W2136825556 @default.
• W2072804535 cites W2138447190 @default.
• W2072804535 cites W2147114673 @default.
• W2072804535 cites W2789682581 @default.
• W2072804535 cites W3006276254 @default.
• W2072804535 cites W4238706447 @default.
• W2072804535 doi "https://doi.org/10.1038/sj.emboj.7600251" @default.
• W2072804535 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/449766" @default.
• W2072804535 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15192701" @default.
• W2072804535 hasPublicationYear "2004" @default.
• W2072804535 type Work @default.
• W2072804535 sameAs 2072804535 @default.
• W2072804535 citedByCount "253" @default.
• W2072804535 countsByYear W20728045352012 @default.
• W2072804535 countsByYear W20728045352013 @default.
• W2072804535 countsByYear W20728045352014 @default.
• W2072804535 countsByYear W20728045352015 @default.
• W2072804535 countsByYear W20728045352016 @default.
• W2072804535 countsByYear W20728045352017 @default.
• W2072804535 countsByYear W20728045352018 @default.
• W2072804535 countsByYear W20728045352019 @default.
• W2072804535 countsByYear W20728045352020 @default.
• W2072804535 countsByYear W20728045352021 @default.
• W2072804535 countsByYear W20728045352022 @default.
• W2072804535 countsByYear W20728045352023 @default.
• W2072804535 crossrefType "journal-article" @default.
• W2072804535 hasAuthorship W2072804535A5001425284 @default.
• W2072804535 hasAuthorship W2072804535A5028711885 @default.
• W2072804535 hasAuthorship W2072804535A5036668859 @default.
• W2072804535 hasAuthorship W2072804535A5051455921 @default.
• W2072804535 hasAuthorship W2072804535A5056187285 @default.
• W2072804535 hasAuthorship W2072804535A5060018103 @default.
• W2072804535 hasAuthorship W2072804535A5068723335 @default.
• W2072804535 hasAuthorship W2072804535A5084260138 @default.
• W2072804535 hasBestOaLocation W20728045351 @default.
• W2072804535 hasConcept C104317684 @default.
• W2072804535 hasConcept C11960822 @default.
• W2072804535 hasConcept C182996813 @default.
• W2072804535 hasConcept C501734568 @default.
• W2072804535 hasConcept C54355233 @default.
• W2072804535 hasConcept C86803240 @default.
• W2072804535 hasConcept C95444343 @default.
• W2072804535 hasConceptScore W2072804535C104317684 @default.
• W2072804535 hasConceptScore W2072804535C11960822 @default.
• W2072804535 hasConceptScore W2072804535C182996813 @default.
• W2072804535 hasConceptScore W2072804535C501734568 @default.

• next