In a recent study, we reported that in bovine brain extract, glycogen synthase kinase-3␤ and tau are parts of an ϳ400 -500 kDa microtubule-associated tau phosphorylation complex (Sun, W., Qureshi, H. Y., Cafferty, P. W., Sobue, K., Agarwal-Mawal, A., Neufield, K. D., and Paudel, H. K. (2002) J. Biol. Chem. 277, 11933-11940). In this study, we find that when purified brain microtubules are subjected to Superose 12 gel filtration column chromatography, the dimeric scaffold protein 14-3-3 coelutes with the tau phosphorylation complex components tau and GSK3␤. From gel filtration fractions containing the tau phosphorylation complex, 14-3-3, GSK3␤, and tau co-immunoprecipitate with each other. From extracts of bovine brain, COS-7 cells, and HEK-293 cells transfected with GSK3␤, 14-3-3 co-precipitates with GSK3␤, indicating that GSK3␤ binds to 14-3-3. From HEK-293 cells transfected with tau, GSK3␤, and 14-3-3 in different combinations, tau co-immunoprecipitates with GSK3␤ only in the presence of 14-3-3. In vitro, ϳ10-fold more tau binds to GSK3␤ in the presence of than in the absence of 14-3-3. In transfected HEK-293 cells, 14-3-3 stimulates GSK3␤-catalyzed tau phosphorylation in a dose-dependent manner. These data indicate that in brain, the 14-3-3 dimer simultaneously binds and bridges tau and GSK3␤ and stimulates GSK3␤-catalyzed tau phosphorylation.

In Alzheimer's disease, microtubule-associated protein tau is hyperphosphorylated by an unknown mechanism and is aggregated into paired helical filaments. Hyperphosphorylation causes loss of tau function, microtubule instability, and neurodegeneration. Glycogen synthase kinase-3␤ (GSK3␤) has been implicated in the phosphorylation of tau in normal and Alzheimer's disease brain. The molecular mechanism of GSK3␤-tau interaction has not been clarified. In this study, we find that when microtubules are disassembled, microtubuleassociated GSK3␤ dissociates from microtubules. From a gel filtration column, the dissociated GSK3␤ elutes as an ϳ400-kDa complex. When fractions containing the ϳ400-kDa complex are chromatographed through an anti-GSK3␤ immunoaffinity column, tau co-elutes with GSK3␤. From fractions containing the ϳ400-kDa complex, both tau and GSK3␤ co-immunoprecipitate with each other. GSK3␤ binds to nonphosphorylated tau, and the GSK3␤-binding region is located within the N-terminal projection domain of tau. In vitro, GSK3␤ associates with microtubules only in the presence of tau. From brain extract, ϳ6-fold more GSK3␤ co-immunoprecipitates with tau than GSK3␣. These data indicate that, in brain, GSK3␤ is bound to tau within a ϳ400-kDa microtubule-associated complex, and GSK3␤ associates with microtubules via tau.

In the preceding paper, we showed that GSK3beta phosphorylates tau at S(202), T(231), S(396), and S(400) in vivo. Phosphorylation of S(202) occurs without priming. Phosphorylation of T(231), on the other hand, requires priming phosphorylation of S(235). Similarly, priming phosphorylation of S(404) is essential for the sequential phosphorylation of S(400) and S(396) by GSK3beta. The priming kinase that phosphorylates tau at S(235) and S(404) in the brain is not known. In this study, we find that in HEK-293 cells cotransfected with tau, GSK3beta, and Cdk5, Cdk5 phosphorylates tau at S(202), S(235), and S(404). S(235) phosphorylation enhances GSK3beta-catalyzed T(231) phosphorylation. Similarly, Cdk5 by phosphorylating S(404) stimulates phosphorylation of S(400) and S(396) by GSK3beta. These data indicate that Cdk5 primes tau for GSK3beta in intact cells. To evaluate if Cdk5 primes tau for GSK3beta in mammalian brain, we examined localizations of Cdk5, tau, and GSK3beta in rat brain. We also analyzed the interaction of Cdk5 with tau and GSK3beta in brain microtubules. We found that Cdk5, GSK3beta, and tau are virtually colocalized in rat brain cortex. When bovine brain microtubules are analyzed by FPLC gel filtration, Cdk5, GSK3beta, and tau coelute within an approximately 450 kDa complex. From the fractions containing the approximately 450 kDa complex, tau, Cdk5, and GSK3beta co-immunoprecipitate with each other. In HEK-293 cells transfected with tau, Cdk5, and GSK3beta in different combinations, tau binds to Cdk5 in a manner independent of GSK3beta and to GSK3beta in a manner independent of Cdk5. However, Cdk5 and GSK3beta bind to each other only in the presence of tau, suggesting that tau connects Cdk5 and GSK3beta. Our results suggest that in the brain, tau, Cdk5, and GSK3beta are components of an approximately 450 kDa complex. Within the complex, Cdk5 phosphorylates tau at S(235) and primes it for phosphorylation of T(231) by GSK3beta. Similarly, Cdk5 by phosphorylating tau at S(404) primes tau for a sequential phosphorylation of S(400) and S(396) by GSK3beta.

Alzheimer's disease (AD) is characterized by the presence of abnormal, straight filaments and paired helical filaments (PHFs) that are coated with amorphous aggregates. When PHFs are treated with alkali, they untwist and form filaments with a ribbonlike morphology. Tau protein is the major component of all of these ultrastructures. 14-3-3ζ is present in NFTs and is significantly upregulated in AD brain. The molecular basis of the association of 14-3-3ζ within NFTs and the pathological significance of its association are not known. In this study, we have found that 14-3-3ζ is copurified and co-immunoprecipitates with tau from NFTs of AD brain extract. In vitro, tau binds to both phosphorylated and nonphosphorylated tau. When incubated with 14-3-3ζ, tau forms amorphous aggregates, single-stranded, straight filaments, ribbonlike filaments, and PHF-like filaments, all of which resemble the corresponding ultrastructures found in AD brain. Immuno-electron microscopy determined that both tau and 14-3-3ζ are present in these ultrastructures and that they are formed in an incubation time-dependent manner. Amorphous aggregates are formed first. As the incubation time increases, the size of amorphous aggregates increases and they are incorporated into single-stranded filaments. Single-stranded filaments laterally associate to form double-stranded, ribbonlike, and PHF-like filaments. Both tau and phosphorylated tau aggregate in a similar manner when they are incubated with 14-3-3ζ. Our data suggest that 14-3-3ζ has a role in the fibrillization of tau in AD brain, and that tau phosphorylation does not affect 14-3-3ζ-induced tau aggregation.

In Parkinson disease (PD) brain, a progressive loss of dopaminergic neurons leads to dopamine depletion in the striatum and reduced motor function. Lewy bodies, the characteristic neuropathological lesions found in the brain of PD patients, are composed mainly of ␣-synuclein protein.

Transforming growth factor (TGF-beta1) is a potent inducer of chondrogenesis and stimulant of cartilage extracellular matrix (ECM) synthesis. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is located in ECM and is the major inhibitor of matrix metalloproteinases (MMPs) and aggrecanase, the principal enzymes implicated in collagen and aggrecan degradation in arthritis. We investigated the role of extracellular-signal-regulated kinase (ERK)-mitogen-activated protein kinases (MAPK) and Sp1 transcription factor in TGF-beta-induced TIMP-3 gene in chondrocytes and chondrosarcoma cells. TGF-beta time-dependently induced a sustained phosphorylation of ERK-MAPKs in primary human or bovine chondrocytes. Inhibitors of this pathway, PD98059 and U0126, downregulated TGF-beta-induced expression of TIMP-3 RNA and protein. Since the ERKs can phosphorylate Sp1, and the promoter of human TIMP-3 gene contains four Sp1-binding sites, we investigated whether Sp1 is a downstream target of this pathway. Mithramycin and WP631, the agents that prevent binding of Sp1 to its consensus site, downregulated TGF-beta-inducible TIMP-3 expression. Indeed, mithramycin blocked TGF-beta-stimulated Sp1 binding activity. Transfection of cytomegalovirus (CMV) promoter-Sp1 plasmid increased TIMP-3 promoter (-940 to +376)-driven luciferase activity. Depletion of Sp1 by transfection of an antisense phosphorothioate oligonucleotide suppressed TGF-beta-induced TIMP-3 protein expression, while its sense homolog had no effect. These results suggest that activation of ERK-MAPK pathway and Sp1 transcription factor play a pivotal role in the induction of TIMP-3 by TGF-beta in chondrocytes.

In the normal brain, tau protein is phosphorylated at a number of proline-and non-proline directed sites, which reduce tau microtubule binding and thus regulate microtubule dynamics. In Alzheimer disease (AD), tau is abnormally hyperphosphorylated, leading to neurofibrillary tangle formation and microtubule disruption, suggesting a loss of regulatory mechanisms controlling tau phosphorylation. Early growth response 1 (Egr-1) is a transcription factor that is significantly up-regulated in AD brain. The pathological significance of this up-regulation is not known. In this study, we found that lentivirus-mediated overexpression of Egr-1 in rat brain hippocampus and primary neurons in culture activates proline-directed kinase Cdk5, inactivates PP1, promotes tau phosphorylation at both proline-directed Ser 396/404 and non-proline-directed Ser 262 sites, and destabilizes microtubules. Furthermore, in Egr-1 ؊/؊ mouse brain, Cdk5 activity was decreased, PP1 activity was increased, and tau phosphorylation was reduced at both proline-directed and non-proline-directed sites. By using nerve growth factorexposed PC12 cells, we determined that Egr-1 activates Cdk5 to promote phosphorylation of tau and inactivates PP1 via phosphorylation. When Cdk5 was inhibited, tau phosphorylation at both proline-and non-proline directed sites and PP1 phosphorylation were blocked, indicating that Egr-1 acts through Cdk5. By using an in vitro kinase assay and HEK-293 cells transfected with tau, PP1, and Cdk5, we found that Cdk5 phosphorylates Ser 396/404 directly. In addition, by phosphorylating and inactivating PP1, Cdk5 promotes tau phosphorylation at Ser 262 indirectly. Our results indicate that Egr-1 is an in vivo regulator of tau phosphorylation and suggest that in AD brain increased levels of Egr-1 aberrantly activate an Egr-1/Cdk5/PP1 pathway, leading to accumulation of hyperphosphorylated tau, thus destabilizing the microtubule cytoskeleton.Alzheimer disease (AD) 3 is the most prevalent form of dementia in the elderly and is characterized by the presence of two characteristic pathological hallmarks: senile plaques and neurofibrillary tangles (NFTs) (1, 2). Senile plaques are composed of ␤-amyloid peptides that are derived from sequential cleavage of the amyloid precursor protein. NFTs are composed mostly of the abnormally hyperphosphorylated microtubuleassociated protein tau (3).Recent studies suggest that ␤-amyloid deposition may occur early and initiate pathological events including tau phosphorylation (4). However, it has been shown that tau dysfunction alone is sufficient to cause neurodegeneration and that tau may be required for ␤-amyloid neurotoxicity (5, 6). Tau hyperphosphorylation is an important process in AD neuropathology. In a normal brain, tau binds to and stabilizes the microtubule cytoskeleton. When phosphorylated, however, it is less able to bind to microtubules, leading to microtubule instability. In AD brain, tau hyperphosphorylation destabilizes microtubules, causing cytoskeletal dysfunction and perhaps NFT form...

Transforming growth factor beta (TGF-beta1) promotes cartilage matrix synthesis and induces tissue inhibitor of metalloproteinases-3 (TIMP-3), which inhibits matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme implicated in articular cartilage degradation and joint inflammation. TGF-beta1 activates Akt, ERK and Smad2 pathways in chondrocytes. Here we investigated previously unexplored roles of specific Smads in TGF-beta1 induction of TIMP-3 gene by pharmacological and genetic knockdown approaches. TGF-beta1-induced Smad2 phosphorylation and TIMP-3 protein expression could be inhibited by the Smad2/3 phosphorylation inhibitors, PD169316 and SB203580 and by Smad2-specific siRNA. Specific inhibitor of Smad3 (SIS3) and Smad3 siRNA abolished TGF-beta induction of TIMP-3. Smad2/3 siRNAs also down regulated TIMP-3 promoter-driven luciferase activities, suggesting transcriptional regulation. SiRNA-driven co-Smad4 knockdown abrogated TIMP-3 augmentation by TGF-beta. TIMP-3 promoter deletion analysis revealed that -828 deletion retains the original promoter activity while -333 and -167 deletions display somewhat reduced activity suggesting that most of the TGF-beta-responsive, cis-acting elements are found in the -333 fragment. Chromatin Immunoprecipitation (ChIP) analysis confirmed binding of Smad2 and Smad4 with the -940 and -333 promoter sequences. These results suggest that receptor-activated Smad2 and Smad3 and co-Smad4 critically mediate TGF-beta-stimulated TIMP-3 expression in human chondrocytes and TIMP-3 gene is a target of Smad signaling pathway.