I= STUDIES ON MECHANISM OF TAU PROTEIN HYPERPHOSPHORYLATION IN ALZHEIMER’S DISEASE
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Title of Thesis
STUDIES ON MECHANISM OF TAU PROTEIN HYPERPHOSPHORYLATION IN ALZHEIMER’S DISEASE

Author(s)
Hamid Yaqoob
Institute/University/Department Details
University Of Agriculture /Department Of Chemistry
Session
2004
Subject
Chemistry
Number of Pages
94
Keywords (Extracted from title, table of contents and abstract of thesis)
gsk3β, microtubule, dna, protein, peptide concentrations, phosphorylation

Abstract
In Alzheimer’s disease (AD) microtubule-associated protein tau becomes hyperphosphorylated and aggregates into paired helical filaments. Abnormal phosphorylation has been suggested to cause the loss of tau function, microtubule instability, and neurodegeneration in AD brain. As the GSK3β and 14-3-3c are associated with tau in brain and are considered to be involved in tau hyperphophorylation, so it is necessary to understand their iteraction in brain. To investigate the biochemical nature of microtubule associated GSK3β, 14-3-3c and to study its interaction with tau, the microtubules was dissociated by cold incubationand removed tubulin from the MAP fraction containing GSK3β and 14-3-3c by phosphocellulose chromatography. When the MAP fraction was subjected to an FPLC gel filtration analysis, GSK3β and 14-3-3c eluted as a ~400 kDa complex. 14-3-3c and the tau phosphorylation complex in the microtubule fraction cannot be separated from each other by Phosphocellulose, gel filtration, and Mono S chromatographies. In vitro, 14-3-3c binds to tau and GSK3β. When the ~400-kDa comlex was chromatographed through an anti- GSK3β immunoaffinity column, tau co-eluted with GSK3β. Similarly, tau and GSK3β co-immunoprecipitated with each other from fractions containing the ~400-kDa complex In vitro, GSK3β bound to R-tau. These data indicate that GSK3β, 14-3-3c and tau ae the components of a ~400-kDa microtubule-associated complex.

In this study it is demonstrated that GSK3β binds to microtubules through tau and also determined that GSK3β binds to the N-terminal projection domain of tau. Since the microtubule-binding region of tau does not overlap with the N-terminatlprojection domain, it is likely that tau can simultaneously bind to microtubules and GSK3β in vitro and perhaps in vivo. It was found that tau within brain extract and 400-kDa complex is heterogeneous by using antibodies Tau-1, 12E8, PHF-1, and AD-2 but only Tau-1 cross reacts with the tau that elutes from the anti- GSK3β immunoaffinity column. These data indicate that tau isoforms within GSK3β tau complex are not phosphorylated on Ser198, Ser199, Ser202, Ser298, Ser400, and Ser404 and suggest that GSK3β bound tau isoforms may be in nonphosphorylated states. However in vivo, tau is also phosphorylated on several other sites for which antibodies are not available. Thus, it concludes that within the GSK3β tau complex, tau isoforms are not phosphorylated on above indicated sites. The presence of tau not phosphorylated on Ser198, Ser199, Ser202, Ser235, Ser398, and Ser404 within the tau. GSK3β complex suggests that GSK3β tau interaction may be regulated by phosphorylation of tau on these sites. Since Cdk5 phosphorylates Ser199, Ser202, Ser235, Ser398, and Ser404 in vitro. GSK3β binds to R-tau but not Cdk5-phosphorylated R-tau. Previous studies have shown that Cdk5 is also a component of neuronal microtubules, binds to tau, and associates with microtubules via tau in a manner similar to GSK3β. Taken together, these observations suggest that GSK3β tau binding may be regulated by phosphorylation of tau by Cdk5. This in turn may suggest that Cdk5 may not only prime tau for GSK3β action but may also regulate tau- GSK3β binding in vitro and possibly in vivio. GSK3β-tau and tau-microtubule interactions may therefore be regulated by phosphorylation on the same tau sites. It is possible that, in the brain, nonphosphorylated tau simultaneously binds to GSK3β and microtubules, bridges GSK3β tomicrotubules, and stabilizes microtubule structure. Upon phosphorylation, tau dissociates from both microtubules and GSK3β.

It was observed that GSK3β and GSK3β are stably associated with microtubules in bovine brain extact. Despite the amounts of GSK3a and GSK3β in brain extract and MAP fraction being almost equal, ~6-fold more GSK3β co-immunoprecipitates with tau from brain extract and MAP fraction than GSK3a indicating that~6-fold more tau is complexed with GSK3β than with GSK3a in brain. With such a profound difference between the amounts of each kinase comnplexed with tau, it is very likely that most of the tau in brain will be phosphorylated by GSK3β than by GSK3a.

This study indicates that GSK3β, tau, and 14-3-3c are parts of a microtubule-associated tau phosphorylation complex. Within the complex, 14-3-3c binds to tau and GSK3β simultaneously and assembles the complex. Thus, the role of 14-3-3c within the tau phosphorylation complex appears to be similar to that of a xin within the β-catenin destruction complex. Furthermore, 14-3-3c binds to tau and changes the tau conformation, making tau susceptible for hyperphosphorylation in vitro and perhaps in vivo. These observations suggest that 14-3-3c not only enhances association of tau and GSK3β within the complex but also prepares tau for GSK3β action.

Download Full Thesis
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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
862.85 KB
2 1 Introduction 01
1092.66 KB
3 2 Review of Literature 07
7075.16 KB
4 3 Materials and Methods 36
3384.79 KB
  3.1 Preparation of microtubule fractions 36
  3.2 Purification of GSK3β from the Microtubules Fraction 37
  3.3 Preparation of Anti-GSK3β Immunoaffinity Column 38
  3.4 GSK3 Activity Assay 38
  3.5 Purification of 14-3-3c from the Microtubules Fraction 39
  3.6 Tau cDNA Cloning 41
  3.7 GSK3β cDNA Cloning 42
  3.8 14-3-3c cDNA cloning 43
  3.9 Proteins 44
  3.10 Protein and Peptide Concentrations 48
5 4 Results 51
3920.5 KB
  Gel Filtration Analysis of Microtubule-associated GSK3 51
  GSK3β Directly Binds to Tau 54
  GSKβ-binding Region within Tau 56
  Effect of Phosphorylation on the Binding of GSK3β to Tau 56
  GSK3β Associates with Microtubules through Tau 58
  Microtubule-associated GSK3a and GSK3β 60
  Microtubule-associated 14-3-3c 64
  Identification of Molecules Bound to 14-3-3c within Brain Microtubules 68
  Binding of 14-3-3c with GSK3β 69
  The role of 14-3-3c in tau phosphorylation 71
6 5 Discussion 73
2098.06 KB
7 6 Summary 82
3366.14 KB
  References 85