14-3-3 Binds to and Mediates Phosphorylation of Microtubule-associated Tau Protein by Ser9-phosphorylated Glycogen Synthase Kinase 3β in the Brain*

In mammalian brain, tau, glycogen synthase kinase 3β (GSK3β), and 14-3-3, a phosphoserine-binding protein, are parts of a multiprotein tau phosphorylation complex. Within the complex, 14-3-3 simultaneously binds to tau and GSK3β (Agarwal-Mawal, A., Qureshi, H. Y., Cafferty, P. W., Yuan, Z., Han, D., Lin, R., and Paudel, H. K. (2003) J. Biol. Chem. 278, 12722–12728). The molecular mechanism by which 14-3-3 connects GSK3β to tau within the complex is not clear. In this study, we find that GSK3β within the tau phosphorylation complex is phosphorylated on Ser9. From extracts of rat brain and rat primary cultured neurons, Ser9-phosphorylated GSK3β precipitates with glutathione-agarose beads coated with glutathione S-transferase-14-3-3. Similarly, from rat brain extract, Ser9-phosphorylated GSK3β co-immunoprecipitates with tau. In vitro, 14-3-3 binds to GSK3β only when the kinase is phosphorylated on Ser9. In transfected HEK-293 cells, 14-3-3 binds to Ser9-phosphorylated GSK3β and does not bind to GSK3β (S9A). Tau, on the other hand, binds to both GSK3β (WT) and GSK3β (S9A). Moreover, 14-3-3 enhances the binding of tau with Ser9-phosphorylated GSK3β by ∼3-fold but not with GSK3β (S9A). Similarly, 14-3-3 stimulates phosphorylation of tau by Ser9-phosphorylated GSK3β but not by GSK3β (S9A). In transfected HEK-293 cells, Ser9 phosphorylation suppresses GSK3β-catalyzed tau phosphorylation in the absence of 14-3-3. In the presence of 14-3-3, however, Ser9-phosphorylated GSK3β remains active and phosphorylates tau. Our data indicate that within the tau phosphorylation complex, 14-3-3 connects Ser9-phosphorylated GSK3β to tau and Ser9-phosphorylated GSK3β phosphorylates tau.

Tau is a neuronal microtubule-associated protein. It is involved in the regulation of microtubule dynamics, axonal transport, and neuronal morphology. In the brain, tau binds to microtubules, promotes microtubule assembly, and stabilizes microtubule structure (for reviews, see Refs. [1][2][3]. Tau is phosphorylated in vivo, and phosphorylation reduces the affinity of tau for microtubules (1,3). Recently, tau has become a center of investigation due to its role in the various neurological disorders collectively called tauopathies, which include Alzheimer's disease (AD) 1 and FTDP-17 (2,4). Tau mutations have been discovered in familial FTDP-17. These mutations cause tau dysfunction and neurodegeneration in cell and animal models (2,4). In AD, abnormally hyperphosphorylated tau (phosphorylated on more sites than normal tau) aggregates and forms paired helical filaments (PHFs) (5,6). PHFs are the main structural component of neurofibrillary tangles, which are one of the characteristic neuropathological lesions found in the brains of patients suffering from AD (2,3). Since abnormally hyperphosphorylated tau, which does not bind to microtubules, regains its biological activity upon dephosphorylation, abnormal hyperphosphorylation has been suggested as being one of the central events in the development of neurofibrillary pathology (2,3). Abnormal tau phosphorylation causes loss of tau function, microtubule instability, and neurodegeneration in AD brain. Preventing or reducing excessive tau phosphorylation has been suggested as a possible therapeutic strategy in AD research (2). A loss in the regulatory mechanism that controls tau phosphorylation in normal brain was suggested to cause abnormal tau phosphorylation in AD brain (2,3). The mechanism by which tau is phosphorylated in the brain is not clear.
Tau is phosphorylated by many kinases in vitro. These kinases include microtubule-associated protein kinase, cAMP-dependent protein kinase, calmodulin-dependent protein kinase 2, cyclin-dependent protein kinase 5, and glycogen synthase kinase 3␤ (GSK3␤) (see Ref. 7 for a list). Biochemical studies have shown that GSK3␤ is physically complexed with tau in the brain and phosphorylates tau in vitro (8,9) and in vivo (10 -13). GSK3␤ is a major tau kinase in the brain (8).
Recently, we found that GSK3␤ and tau are parts of a ϳ500-kDa multiprotein tau phosphorylation complex (8). More recently, we reported 14-3-3 as the third component of the complex (9). Within the complex, 14-3-3 binds to GSK3␤, connects it to tau, and stimulates GSK3␤-catalyzed tau phosphorylation (9). The biochemical mechanism by which 14-3-3 interacts with GSK3␤ is not known. A characteristic feature of 14-3-3 is that it binds to a phosphorylated serine within its target protein (27)(28)(29)(30)(31). Studies based on synthetic peptides derived from Raf suggest that the 14-3-3-binding region consists of a short stretch of amino acid residues containing a phosphoserine (27,30). Within this sequence, an Arg residue at position Ϫ3 relative to the phosphoserine was suggested to be critical for 14-3-3 binding (30). GSK3␤ has an Arg at position Ϫ3 relative to Ser 9 (16). Many 14-3-3-binding proteins contain a Ser or a Thr at position Ϫ1 and/or Ϫ2 with respect to phosphoserine involved in 14-3-3 binding (see Ref. 27 for a list). GSK3␤ contains a Thr at both positions Ϫ1 and Ϫ2 with respect to Ser 9 (16). Like BAD, tyrosine phosphatase PTH1, and Wee1, which contain a hydrophobic residue at position ϩ1 with respect to their 14-3-3-binding phosphoserine (32)(33)(34), GSK3␤ contains a Phe residue at position ϩ1 with respect to Ser 9 (16). These observations suggest that GSK3␤ contains a 14-3-3-binding sequence around its Ser 9 . Since GSK3␤ is phosphorylated on Ser 9 in vivo (19 -21), we have investigated the interaction of 14-3-3 and GSK3␤ within the tau phosphorylation complex. Herein, we report that 14-3-3 binds to phosphorylated Ser 9 of GSK3␤ in the brain. We also show that 14-3-3 connects Ser 9 -phosphorylated GSK3␤ to tau and facilitates tau phosphorylation by Ser 9phosphorylated GSK3␤. Our results suggest a novel mechanism that explains how GSK3␤, upon phosphorylation of Ser 9 , in response to various growth factors, may phosphorylate tau in the brain (24 -26).
Cell Culture and Transfection-To culture primary neurons, 15 fresh brains of 1-day postnatal rat pups were cut into small pieces and then transferred into a 50-ml Falcon tube containing 30 ml of PBS (25 mM Na 2 HPO 4 (pH 7.4), 137 mM NaCl). After adding 3 ml of trypsin (2.5%), the tube was incubated for 15 min at 37°C with occasional mixing by tube inversion. To the incubated mixture, 3 ml of fetal bovine serum (HyClone, Logan, UT) followed by 4 ml of DNase I (1 mg/ml) were added. The tube was subjected to five or six inversions followed by gentle trituration using a 25-ml glass pipette until the mixture became homogeneous. The mixture was filtered twice through a nylon membrane. The neurons in the filtrate were pelleted by centrifugation for 10 min at 10°C. The pellet was washed twice with PBS and then dispersed in 45 ml of culture medium (minimum essential medium (Invitrogen) supplemented with 30 mM glucose, 2 mM glutamine, 1 mM pyruvate, and 10% fetal bovine serum). Neurons in the dispersed solution were plated at 3 ϫ 10 6 cells/mm in 35-mm poly-D-lysine-coated dishes and placed in a CO 2 incubator maintained at 37°C. The culture medium was replaced with fresh culture medium supplemented with 1 mM fluorodeoxyuridine (Sigma) on the second and the third days. On the fourth day, the culture medium was again replaced with fresh culture medium without fluorodeoxyuridine. On the seventh day, neurons were scraped and suspended in 0.5 ml of lysis buffer (50 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 25 mM ␤-glycerophosphate, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 10 mM MgCl 2 , 0.1% Nonidet P-40, 25 nM okadaic acid, 4 nM cypermethrin, 1 mM phenylmethylsulfonyl fluoride, and 2 g/ml each of leupeptin, pepstatin, and aprotinin). The suspension was incubated on ice with occasional shaking for 30 min and centrifuged, and the supernatant was used for a GST pull-down assay.
HEK-293 cells maintained in Dulbecco's modified high glucose Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum were plated on 10-cm dishes. Once ϳ80% confluent, cells were transfected with 0.2-10 g of cDNA using LipofectAMINE Plus reagent (Invitrogen) following the manufacturer's instructions. The amount of DNA used for each transfection in various experiments was as follows: 1 g of tau, 1 g of HA-GSK3␤ (WT), 3 g of HA-GSK3␤ (S9A), 6 g of HA-AKT, and 0.2 g of Myc-14-3-3. For each transfection, the amount of total DNA never exceeded 10 g. Cells were harvested 72 h after transfection and used for GST pull-down assays, immunoblottings, and immunoprecipitations.
Monoclonal and polyclonal antibodies specific for GSK3␤ phosphorylated on Ser 9 and GSK3␣ phosphorylated on Ser 21 were purchased from Upstate Biotechnology and MBA International Corp. (Watertown, MA), respectively. Monoclonal antibodies, anti-HA, anti-GSK3␤, antitau, and PHF-1, are described previously (9). Polyclonal antibodies against tau and GSK3␤ used to immunoprecipitate respective proteins have also been described previously (8). Anti-Myc monoclonal antibody was from Cell Signaling Technology Inc. (Beverly, MA).
Immunoprecipitation-HEK-293 cells transfected with various genes were washed with cold phosphate-buffered saline, scraped, and suspended in 0.8 ml of cold lysis buffer. The suspension was incubated on ice for ϳ30 min with occasional gentle shaking and then centrifuged at full speed using a bench top centrifuge at 4°C. The supernatant was used for immunoprecipitation, GST pull-down assay, and immunoblotting. The procedure for immunoprecipitation is essentially as described (9).
To generate Figs. 1 and 7, B-D, 4.5 g of fresh rat brain was cut into small pieces and then homogenized in 4 ml of lysis buffer using a glass homogenizer. The resulting homogenate was centrifuged at 27,000 ϫ g for 40 min at 4°C. The supernatant was used for immunoprecipitation using the indicated antibodies as described (8).
GST Pull-down Assay-Brain extract or cell lysate (0.2 ml each) were mixed with 50 l of glutathione-agarose beads (Sigma) coated with GST or GST-14-3-3 and then shaken end-over-end for 3 h at 4°C. The mixture was then centrifuged, and the pellet was washed three times with the lysis buffer, mixed with 50 l of SDS-PAGE sample buffer, boiled, and centrifuged, and 10 l of supernatant was analyzed by SDS-PAGE or immunoblot analysis using the indicated antibodies.
To determine whether the above observation is due to 14-3-3-bound GSK3␤ being phosphorylated on Ser 9 or due to a nonspecific interaction of the antibody used to generate Fig.  1C, we repeated the above experiment using a polyclonal antibody specific for GSK3␤ phosphorylated on Ser 9 and GSK3␣ phosphorylated on Ser 21 . Again, GSK3␤ that came down with glutathione-agarose beads coated with GST-14-3-3 displayed immunoreactivity (data not shown). To substantiate the above observations, we mixed glutathione-agarose beads coated with GST or GST-14-3-3 with fresh lysates from cultured primary rat neurons. Beads were washed and then immunoblotted with anti-GSK3␤ or anti-GSK3␤-pS9 antibody. As expected, GSK3␤ specifically bound to GST-14-3-3 and was phosphorylated on Ser 9 (data not shown). These observations suggested that 14-3-3 binds to Ser 9 -phosphorylated GSK3␤ in the brain.

FIG. 2. Ser 9 to Ala mutant of GSK3␤ does not bind to 14-3-3.
HEK-293 cells transfected with the indicated constructs were lysed, and each lysate was subjected to GST pull-down assay or co-immunoprecipitation. A, GST pull-down assay. Glutathione-agarose beads coated with GST or GST-14-3-3 were incubated with lysates of cells transfected with either HA-GSK3␤ (WT) or HA-GSK3␤ (S9A). Beads were washed and immunoblotted (IB) with anti-HA antibody. Both HA-GSK3␤ (WT) and HA-GSK3␤ (S9A) migrate with similar sizes on a SDS-gel, are HA-tagged, and are recognized by anti-HA antibody. B and C, co-immunoprecipitation. HA-GSK3␤ (WT) and HA-GSK3␤ (S9A) were immunoprecipitated (IP) from respective cell lysates using anti-HA antibody. Each immune complex was then immunoblotted with the indicated antibody. D and E, immunoblots. Cell lysates used to generate B and C were immunoblotted with the indicated antibodies to monitor expression of the indicated genes. This experiment was repeated at least four times with similar results. . These observations confirm that 14-3-3 binds to HA-GSK3␤ (WT) but not HA-GSK3␤ (S9A). Thus, mutation of Ser 9 of GSK3␤ to Ala completely abolishes binding of GSK3␤ with 14-3-3.
Tau-associated GSK3␤ in the Brain Is Ser 9 -phosphorylated-In the brain, tau, GSK3␤, and 14-3-3 are parts of a multiprotein tau phosphorylation complex (9). Within the complex, 14-3-3 binds to GSK3␤ (9). Since we find that 14-3-3 associates with Ser 9 -phosphorylated GSK3␤ in the brain (Fig.  1), we investigated whether GSK3␤ within the tau phosphorylation complex is also Ser 9 -phosphorylated. We analyzed a partially purified tau phosphorylation complex preparation from  Fig. 3 were incubated with glutathioneagarose beads coated with GST or GST-14-3-3. Beads were washed and immunoblotted (IB) with anti-HA antibody to detect HA-GSK3␤. This experiment was repeated at least three times with similar results.
The Interaction of Tau with GSK3␤ (S9A) Is Insensitive to 14-3-3-Previous studies have shown that although tau binds directly to the N-terminal region of GSK3␤, this binding is weak (8,9). 14-3-3 simultaneously binds to tau and GSK3␤ and enhances the association of the two proteins (9). Therefore, in the presence of 14-3-3, GSK3␤ strongly binds to tau. In this study, we find that 14-3-3 binds to Ser 9 -phosphorylated GSK3␤. Therefore, it may be possible that 14-3-3 bridges Ser 9phosphorylated GSK3␤ to tau, and this may be why GSK3␤ within the tau phosphorylation complex is Ser 9 -phosphorylated (Fig. 7). If so, 14-3-3 should not be able to enhance the association of GSK3␤ (S9A) with tau.
To test the above idea, we transfected HEK-293 cells with tau, Myc-14-3-3, and HA-GSK3␤ or HA-GSK3␤ (S9A). Transfected cells were lysed and subjected to immunoprecipitation using anti-HA antibody. This antibody effectively immunopre- FIG. 5. Phosphorylation enhances the interaction of GSK3␤ with 14-3-3. Lysates of HEK-293 cells transfected with the indicated constructs were either immunoblotted (IB) to analyze GSK3␤ phosphorylation and expression of various genes or used to assay binding of Myc-14-3-3 to HA-GSK3␤ by co-immunoprecipitation. Blots corresponding to HA-GSK3␤ and Myc-14-3-3 were scanned, and band intensity values of various bands were obtained and used to determine the fraction of GSK3␤ phosphorylated or fraction of 14-3-3 bound to HA-GSK3␤. A, HA-GSK3␤ phosphorylation on Ser 9 . B-D, expression of various genes. E, co-immunoprecipitation (IP). GSK3␤ was immunoprecipitated from indicated lysates, and the resulting immune complex was immunoblotted with anti-Myc antibody. Note that both HA-GSK3␤ and HA-AKT are immunoreactive to anti-HA antibody. Therefore, we have used anti-GSK3␤ polyclonal antibody to immunoprecipitate HA-GSK3␤ in this experiment. F, a comparison between the relative fraction of HA-GSK3␤ phosphorylated and the relative fraction of 14-3-3 bound to HA-GSK3␤. To determine the relative fraction of GSK3␤ phosphorylated on Ser 9 , band intensity values of lanes 3 and 4 from A representing GSK3␤ phosphorylation were divided by respective band intensity values from B representing total GSK3␤. Each resulting value was then divided by the resulting value for lane 3. Similarly, to determine the relative fraction of Myc-14-3-3 bound to HA-GSK3␤, band intensity values of lanes 3 and 4 from E representing 14-3-3 co-immunoprecipitating with HA-GSK3␤ were divided by respective band intensity values from D, representing total Myc-14-3-3. Each resulting value for lanes 3 and 4 was then divided by the resulting value for lane 3. The values with S.E. represent three independent experiments.
Phosphorylation of tau in cells transfected with tau and HA-GSK3␤ (S9A) was higher than that in cells transfected with tau alone (Fig. 10, A, lane 5, and E), indicating that GSK3␤ (S9A) phosphorylates tau in intact cells. However, the amount of tau phosphorylation was not significantly different between cells transfected with tau plus HA-GSK3␤ (S9A) and those transfected with tau plus HA-GSK3␤ (S9A) and Myc-14-3-3 (compare lanes 5 and 7 in Fig. 10A, and see Fig. 10E). These observations indicate that 14-3-3 does not influence tau phosphorylation by GSK3␤ (S9A). FIG. 7. Tau-associated GSK3␤ in the brain is Ser 9 -phosphorylated. To examine whether tau bound GSK3␤ in the brain is Ser 9 -phosphorylated, we either analyzed a partially purified tau phosphorylation complex from bovine brain extract by FPLC gel filtration or performed a co-immunoprecipitation. A, FPLC gel filtration. Gel filtration was carried out at 4°C using an Amersham Biosciences FPLC system and a FPLC Superose 12 gel filtration column (50 ϫ 1.6 cm) equilibrated in 25 mM MOPS (pH 7.2), 0.1 mM EDTA, 0.1 mM DTT, 200 mM NaCl, 50 mM ␤-glycerophosphate, and 10 mM NaF. Fractions (1 ml each) were collected, and aliquots (15 l each) from the indicated fractions were used for GSK3 activity assay (8) or immunoblot analysis using the indicated antibodies. B-D, co-immunoprecipitation. A fresh rat brain extract was subjected to immunoprecipitation using the indicated antibodies. Each resulting immune complex was then immunoblotted with the indicated antibody. Similar observations were made in at least four independent experiments. Phosphorylation of Tau by Ser 9 -phosphorylated GSK3␤-Ser 9 phosphorylation has been suggested to down-regulate GSK3␤ activity (17,18). However, our data suggest that 14-3-3 facilitates tau phosphorylation by Ser 9 -phosphorylated GSK3␤. Therefore, to examine how phosphorylation of GSK3␤ on Ser 9 affects tau phosphorylation by this kinase, we transfected HEK-293 cells with HA-GSK3␤, tau, HA-AKT, and Myc-14-3-3 in various combinations. Transfected cells were lysed, and cell lysates were evaluated for both phosphorylation of GSK3␤ on Ser 9 and tau phosphorylation. Based on tau band intensities, the relative amounts of tau phosphorylated in various transfected cells were determined.
In various cell types, AKT phosphorylates GSK3␤ on Ser 9 but causes only partial inhibition of GSK3␤ activity. For example, in L8 myotubes, insulin stimulates AKT by more than 10-fold and causes phosphorylation of 60 -100% of total GSK3␤ on Ser 9 , and only 40 -50% of total GSK3␤ activity is inhibited (20). In NIH 3T3 cells, GSK3␤ is highly phosphorylated on Ser 9 when cells are exposed to epidermal growth factor with only a 50% drop in GSK3␤ activity (38). In A431 cells, epidermal growth factor, which is known to stimulate AKT and phosphorylation of GSK3␤ on Ser 9 (38), activates GSK3␤ (39). Several other studies have reported similar results in various cell lines (38 -40). Thus, although in vitro Ser 9 -phosphorylated GSK3␤ is inactive (17, 19, 22), a pool of Ser 9 -phosphorylated GSK3␤ in vivo remains active (20, 38 -41). These observations suggest that Ser 9 phosphorylation of GSK3␤ has a role other than inhibiting kinase activity and that there is a mechanism to maintain GSK3␤ active upon its phosphorylation on Ser 9 .
GSK3␤ is a component of cell signaling pathways mediated by various growth factors including insulin, insulin-like growth factor (IGF), and fibroblast growth factor (FGF). These growth factors activate AKT, which then phosphorylates GSK3␤ on Ser 9 (15,20,21). Mammalian brain is rich in insulin, IGF, and FGF receptors, and studies indicate that tau phosphorylation is regulated by these growth factors in mature and developing brains as well as in differentiating neurons in culture (21, 24 -26, 42). When human neuroblastoma cells or rat cortical neurons are treated with insulin or IGF, GSK3␤ phosphorylates tau (24,25). Similarly, GSK3␤ phosphorylates tau in neuronal progenitor cells in response to FGF exposure (26). As discussed above, AKT phosphorylates GSK3␤ on Ser 9 when neurons are exposed to insulin, IGF, or FGF (14,15,21,43). These observations suggest that in neurons, GSK3␤ phosphorylates tau upon its phosphorylation on Ser 9 .
Transgenic mice overexpressing GSK3␤ (WT) display upregulated GSK3␤ activity and neurodegeneration (11). Surprisingly, transgenic mice overexpressing GSK3␤ (S9A) show high GSK3␤ activity in their brain extract but develop normally without any neurodegeneration (12) (for commentary, see Ref. 44). More importantly, tau is hyperphosphorylated in the brains of mice overexpressing GSK3␤ (WT) (11). In the brains of transgenic mice overexpressing GSK3␤ (S9A), tau is not phosphorylated significantly even until the age of 5-7 months (12). The reason why GSK3␤ (WT) and GSK3␤ (S9A) have such opposite effects in the brain is not known. However, the sole difference between GSK3␤ (WT) and GSK3␤ (S9A) is that only the former can be phosphorylated on Ser 9 . Thus, despite being constitutively active, GSK3␤ (S9A) is not capable of phosphorylating tau significantly in a manner similar to GSK3␤ (WT) in vivo. Consistent with the view that Ser 9 phosphorylation is important for GSK3␤ to phosphorylate tau in the brain, starvation induces Ser 9 phosphorylation of GSK3␤ with a concomitant increase in GSK3␤-catalyzed tau phosphorylation in the brains of adult mice (45).
This study emphasizes the mechanism by which 14-3-3 promotes binding of Ser 9 -phosphorylated GSK3␤ with tau. In this mechanism, 14-3-3 simultaneously binds to tau and Ser 9 -phosphorylated GSK3␤. Thus, GSK3␤ and tau do not interact di- rectly but thorough 14-3-3. Furthermore, GSK3␤ must be phosphorylated on Ser 9 to be able to bind to 14-3-3. However, in a previous study, using recombinant proteins, we showed that in vitro, 14-3-3 promotes binding of tau with nonphosphorylated GSK3␤ as well (9). This observation indicates that 14-3-3 also promotes binding of tau with GSK3␤ by a mechanism that does not require phosphorylation of GSK3␤ on Ser 9 .
At this moment, we do not know the mechanism by which 14-3-3 enhances binding of GSK3␤ with tau in a Ser 9 phosphorylation-independent manner. However, 14-3-3 binds to GSK3␤ only when GSK3␤ is phosphorylated on Ser 9 (Fig. 6), and therefore 14-3-3 cannot bind and connect GSK3␤ to tau when the kinase is not phosphorylated on Ser 9 . Instead, previous studies have shown that GSK3␤, in addition to interacting with tau via 14-3-3, also binds to tau directly with low affinity (8,9). More importantly, this binding does not require GSK3␤ to be phosphorylated on Ser 9 (8). 14-3-3, on the other hand, binds to tau and changes tau conformation (7). Moreover, GSK3␤ and 14-3-3 binding sites within tau do not overlap (7,8). It is therefore possible that 14-3-3 binds to tau and induces a conformational change thereby enhancing the affinity of tau for GSK3␤. As a result, more tau binds to GSK3␤ in the presence of 14-3-3 than in the absence (9). This means that 14-3-3 not only connects Ser 9 -phosphorylated GSK3␤ to tau but also enhances the affinity of tau for GSK3␤. Although this may explain why in vitro more tau binds to GSK3␤ in the presence of 14-3-3 than in the absence (9), we find that the binding of tau with GSK3␤ (S9A) is insensitive to 14-3-3 in intact cells (Fig.  9). These observations suggest that in vivo, 14-3-3 regulates the interaction of tau with GSK3␤ only when the kinase is phosphorylated on Ser 9 and are consistent with the idea that 14-3-3 binds to Ser 9 -phosphorylated GSK3␤ first and then subsequently targets the kinase to tau.
Within the tau phosphorylation complex, 14-3-3 is the central molecule and holds GSK3␤ and tau together (9). As discussed above, 14-3-3 is a phosphoserine-binding protein (27)(28)(29), and GSK3␤ must be phosphorylated on Ser 9 before 14-3-3 can bind to and target this kinase to the tau phosphorylation complex in the brain. GSK3␤ is known to be phosphorylated on Ser 9 in response to various extracellular signals (15,17,18,21,43). It is possible that the tau phosphorylation complex is in dynamic equilibrium regulated by phosphorylation of GSK3␤ on Ser 9 . During growth factor signaling, phosphorylation of GSK3␤ on Ser 9 may signal 14-3-3 to bind to and target Ser 9phosphorylated GSK3␤ to the tau phosphorylation complex and facilitate tau phosphorylation by Ser 9 -phosphorylated FIG. 11. Effect of 14-3-3 on tau phosphorylation by Ser 9 -phosphorylated GSK3␤. HEK-293 cells transfected with the indicated constructs were lysed, and each lysate was analyzed by immunoblot (IB) analysis to evaluate HA-GSK3␤ phosphorylation on Ser 9 , expressions of various genes, tau phosphorylation, and relative amount of tau phosphorylated. A-F, immunoblots. G, relative amount of tau phosphorylated. Blots E and F were scanned, and intensity values of each tau band in lanes 2-9 from F were divided by the intensity value of the corresponding lane from E. Each resulting value was then divided by the resulting value for lane 2. The values are averages of three independent determinations.