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Tau phosphorylation by GSK-3beta promotes tangle-like filament morphology.

Rankin CA, Sun Q, Gamblin TC - Mol Neurodegener (2007)

Bottom Line: These results suggest that phosphorylation of tau by GSK-3beta promotes formation of tangle-like filament morphology.Although the severity of dementia has been found to correlate with the presence of NFTs, there is some question as to the identity of the neurotoxic agents involved.This model system will be beneficial in identifying intermediates or side reaction products that might be neurotoxic.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA. crankin@ku.edu

ABSTRACT

Background: Neurofibrillary tangles (NFTs) are intraneuronal aggregates associated with several neurodegenerative diseases including Alzheimer's disease. These abnormal accumulations are primarily comprised of fibrils of the microtubule-associated protein tau. During the progression of NFT formation, disperse and non-interacting tau fibrils become stable aggregates of tightly packed and intertwined filaments. Although the molecular mechanisms responsible for the conversion of disperse tau filaments into tangles of filaments are not known, it is believed that some of the associated changes in tau observed in Alzheimer's disease, such as phosphorylation, truncation, ubiquitination, glycosylation or nitration, may play a role.

Results: We have investigated the effects of tau phosphorylation by glycogen synthase kinase-3beta (GSK-3beta) on tau filaments in an in vitro model system. We have found that phosphorylation by GSK-3beta is sufficient to cause tau filaments to coalesce into tangle-like aggregates similar to those isolated from Alzheimer's disease brain.

Conclusion: These results suggest that phosphorylation of tau by GSK-3beta promotes formation of tangle-like filament morphology. The in vitro cell-free experiments described here provide a new model system to study mechanisms of NFT development. Although the severity of dementia has been found to correlate with the presence of NFTs, there is some question as to the identity of the neurotoxic agents involved. This model system will be beneficial in identifying intermediates or side reaction products that might be neurotoxic.

No MeSH data available.


Related in: MedlinePlus

Tau phosphorylation by GSK-3B. A) SDS-PAGE analysis of tau protein incubated for 20 h in the absence (lanes 1, 4, 7, 10, and 13) or the presence of 0.006 U GSK-3β per pmol tau (lanes 2, 5, 8, 11, 14) or 0.018 U GSK-3β per pmol tau (lanes 3, 6, 9, 12, and 15). A definite band shift in the migration of phosphorylated tau can be detected with increasing time and kinase concentration. B) The amount of γ-32P incorporation over time using 0.018 U GSK-3β per pmol tau (open circles, right y-axis) is compared to the SDS-PAGE analysis in Panel A (open squares, left y-axis). Lines are drawn through the points to ease comparison. Data represents a single trial.
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Figure 1: Tau phosphorylation by GSK-3B. A) SDS-PAGE analysis of tau protein incubated for 20 h in the absence (lanes 1, 4, 7, 10, and 13) or the presence of 0.006 U GSK-3β per pmol tau (lanes 2, 5, 8, 11, 14) or 0.018 U GSK-3β per pmol tau (lanes 3, 6, 9, 12, and 15). A definite band shift in the migration of phosphorylated tau can be detected with increasing time and kinase concentration. B) The amount of γ-32P incorporation over time using 0.018 U GSK-3β per pmol tau (open circles, right y-axis) is compared to the SDS-PAGE analysis in Panel A (open squares, left y-axis). Lines are drawn through the points to ease comparison. Data represents a single trial.

Mentions: The in vitro phosphorylation of monomeric tau by GSK-3β was dependent on both enzyme concentration and incubation time (Figure 1A). Phosphorylation of tau was detected by an upward shift in mobility upon SDS-PAGE analysis reminiscent of hyper-phosphorylated tau in AD [22]. The band shift represents an SDS-resistant conformational change brought about by phosphorylation rather than a molecular weight increase due to added phosphates [35,36]. The in vitro phosphorylation, seen as an upward band shift, was mostly complete after 20 h incubation (Figure 1A, lanes 13, 14, and 15). With no GSK-3β present (lane 13) the tau monomer migrates approximately as a 74 kDa protein. With a kinase concentration of 0.006 U/pmol tau (lane 14), the presence of multiple bands suggest that tau is not fully phosphorylated. At a kinase concentration of 0.018 U/pmol tau (lanes 3, 6, 9, 12, and 15), band density measurements showed that 8% shifted after 15 minutes, 16% by 30 min, 29% by one hour, 39% by 2 hours and 73% after 20 hours.


Tau phosphorylation by GSK-3beta promotes tangle-like filament morphology.

Rankin CA, Sun Q, Gamblin TC - Mol Neurodegener (2007)

Tau phosphorylation by GSK-3B. A) SDS-PAGE analysis of tau protein incubated for 20 h in the absence (lanes 1, 4, 7, 10, and 13) or the presence of 0.006 U GSK-3β per pmol tau (lanes 2, 5, 8, 11, 14) or 0.018 U GSK-3β per pmol tau (lanes 3, 6, 9, 12, and 15). A definite band shift in the migration of phosphorylated tau can be detected with increasing time and kinase concentration. B) The amount of γ-32P incorporation over time using 0.018 U GSK-3β per pmol tau (open circles, right y-axis) is compared to the SDS-PAGE analysis in Panel A (open squares, left y-axis). Lines are drawn through the points to ease comparison. Data represents a single trial.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1936422&req=5

Figure 1: Tau phosphorylation by GSK-3B. A) SDS-PAGE analysis of tau protein incubated for 20 h in the absence (lanes 1, 4, 7, 10, and 13) or the presence of 0.006 U GSK-3β per pmol tau (lanes 2, 5, 8, 11, 14) or 0.018 U GSK-3β per pmol tau (lanes 3, 6, 9, 12, and 15). A definite band shift in the migration of phosphorylated tau can be detected with increasing time and kinase concentration. B) The amount of γ-32P incorporation over time using 0.018 U GSK-3β per pmol tau (open circles, right y-axis) is compared to the SDS-PAGE analysis in Panel A (open squares, left y-axis). Lines are drawn through the points to ease comparison. Data represents a single trial.
Mentions: The in vitro phosphorylation of monomeric tau by GSK-3β was dependent on both enzyme concentration and incubation time (Figure 1A). Phosphorylation of tau was detected by an upward shift in mobility upon SDS-PAGE analysis reminiscent of hyper-phosphorylated tau in AD [22]. The band shift represents an SDS-resistant conformational change brought about by phosphorylation rather than a molecular weight increase due to added phosphates [35,36]. The in vitro phosphorylation, seen as an upward band shift, was mostly complete after 20 h incubation (Figure 1A, lanes 13, 14, and 15). With no GSK-3β present (lane 13) the tau monomer migrates approximately as a 74 kDa protein. With a kinase concentration of 0.006 U/pmol tau (lane 14), the presence of multiple bands suggest that tau is not fully phosphorylated. At a kinase concentration of 0.018 U/pmol tau (lanes 3, 6, 9, 12, and 15), band density measurements showed that 8% shifted after 15 minutes, 16% by 30 min, 29% by one hour, 39% by 2 hours and 73% after 20 hours.

Bottom Line: These results suggest that phosphorylation of tau by GSK-3beta promotes formation of tangle-like filament morphology.Although the severity of dementia has been found to correlate with the presence of NFTs, there is some question as to the identity of the neurotoxic agents involved.This model system will be beneficial in identifying intermediates or side reaction products that might be neurotoxic.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA. crankin@ku.edu

ABSTRACT

Background: Neurofibrillary tangles (NFTs) are intraneuronal aggregates associated with several neurodegenerative diseases including Alzheimer's disease. These abnormal accumulations are primarily comprised of fibrils of the microtubule-associated protein tau. During the progression of NFT formation, disperse and non-interacting tau fibrils become stable aggregates of tightly packed and intertwined filaments. Although the molecular mechanisms responsible for the conversion of disperse tau filaments into tangles of filaments are not known, it is believed that some of the associated changes in tau observed in Alzheimer's disease, such as phosphorylation, truncation, ubiquitination, glycosylation or nitration, may play a role.

Results: We have investigated the effects of tau phosphorylation by glycogen synthase kinase-3beta (GSK-3beta) on tau filaments in an in vitro model system. We have found that phosphorylation by GSK-3beta is sufficient to cause tau filaments to coalesce into tangle-like aggregates similar to those isolated from Alzheimer's disease brain.

Conclusion: These results suggest that phosphorylation of tau by GSK-3beta promotes formation of tangle-like filament morphology. The in vitro cell-free experiments described here provide a new model system to study mechanisms of NFT development. Although the severity of dementia has been found to correlate with the presence of NFTs, there is some question as to the identity of the neurotoxic agents involved. This model system will be beneficial in identifying intermediates or side reaction products that might be neurotoxic.

No MeSH data available.


Related in: MedlinePlus