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GSK-3beta phosphorylation of functionally distinct tau isoforms has differential, but mild effects.

Voss K, Gamblin TC - Mol Neurodegener (2009)

Bottom Line: We have found that each isoform has a unique microtubule binding and polymerization profile that is altered by GSK-3beta.These results indicate that tau phosphorylation by a single kinase can have isoform specific outcomes.The mild nature of these changes, however, makes it unlikely that differential effects of GSK-3beta phosphorylation on the isoforms are causative in neurodegenerative disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA. gamblin@ku.edu.

ABSTRACT

Background: Tau protein exists as six different isoforms that differ by the inclusion or exclusion of exons 2, 3 and 10. Exon 10 encodes a microtubule binding repeat, thereby resulting in three isoforms with three microtubule binding repeats (3R) and three isoforms that have four microtubule binding repeats (4R). In normal adult brain, the relative amounts of 3R tau and 4R tau are approximately equal. These relative protein levels are preserved in Alzheimer's disease, although in other neurodegenerative tauopathies such as progressive supranuclear palsy, corticobasal degeneration and Pick's disease, the ratio of 3R:4R is frequently altered. Because tau isoforms are not equally involved in these diseases, it is possible that they either have inherently unique characteristics owing to their primary structures or that post-translational modification, such as phosphorylation, differentially affects their properties.

Results: We have determined the effects of phosphorylation by a kinase widely believed to be involved in neurodegenerative processes, glycogen synthase kinase-3beta (GSK-3beta), on the microtubule binding and inducer-initiated polymerization of these isoforms in vitro. We have found that each isoform has a unique microtubule binding and polymerization profile that is altered by GSK-3beta. GSK-3beta phosphorylation had differential effects on the isoforms although there were similarities between isoforms and the effects were generally mild.

Conclusion: These results indicate that tau phosphorylation by a single kinase can have isoform specific outcomes. The mild nature of these changes, however, makes it unlikely that differential effects of GSK-3beta phosphorylation on the isoforms are causative in neurodegenerative disease. Instead, the inherent differences in the isoform interactions themselves and local conditions in the diseased cells are likely the major determinant of isoform involvement in various neurodegenerative disorders.

No MeSH data available.


Related in: MedlinePlus

ARA induction of tau isoform polymerization. 2 μM tau isoforms were incubated with various ARA concentrations (0 μM to 100 μM) for 18 hrs. Polymerization was measured by (A) LLS or (B) ThS fluorescence. Symbols correspond as follows: (open square) 0N3R, (closed square) 0N4R, (open triangle) 1N3R, (closed triangle) 1N4R, (open circle) 2N3R, (closed circle) 2N4R, and (closed diamond) assembly incompetent protein (AIP (I277, 308P)). Data is in arbitrary units and represents an average of 3 trials ± SEM. After 18 hours, ARA induced polymerization reactions were visualized by TEM at 20,000x magnification. Images are as follows: (C) 0N3R, (D) 1N3R, (E) 2N3R, (F) 0N4R, (G) 1N4R, and (H) 2N4R. Each isoform image is representative of polymerized material at 75 μM ARA. Scale bars represent 1 μm.
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Figure 2: ARA induction of tau isoform polymerization. 2 μM tau isoforms were incubated with various ARA concentrations (0 μM to 100 μM) for 18 hrs. Polymerization was measured by (A) LLS or (B) ThS fluorescence. Symbols correspond as follows: (open square) 0N3R, (closed square) 0N4R, (open triangle) 1N3R, (closed triangle) 1N4R, (open circle) 2N3R, (closed circle) 2N4R, and (closed diamond) assembly incompetent protein (AIP (I277, 308P)). Data is in arbitrary units and represents an average of 3 trials ± SEM. After 18 hours, ARA induced polymerization reactions were visualized by TEM at 20,000x magnification. Images are as follows: (C) 0N3R, (D) 1N3R, (E) 2N3R, (F) 0N4R, (G) 1N4R, and (H) 2N4R. Each isoform image is representative of polymerized material at 75 μM ARA. Scale bars represent 1 μm.

Mentions: Because the primary sequences of the tau isoforms are different, the various combinations of exons could influence their interactions with inducer molecules or cause conformational changes affecting tau-tau interactions during the polymerization process. The polymerization characteristics of each isoform was determined by incubating 2 μM protein with a range of ARA concentrations (0–150 μM) and monitoring the extent of polymerization with right angle laser light scattering (LLS, Figure 2a) and thioflavine S fluorescence (ThS, Figure 2b). We determined that above 100 μM ARA, the results were highly variable and not easily reproducible (data not shown). This is consistent with previously published results showing high variability at the inhibitory ratio of inducer to 2N4R tau [7]. The isoforms followed the same general trend of increasing amounts of polymerization with increasing inducer concentrations. 4R tau isoforms (containing exon 10) polymerized to a greater extent than 3R isoforms (Figure 2a & 2b), indicating that the additional microtubule binding repeat enhances tau polymerization.


GSK-3beta phosphorylation of functionally distinct tau isoforms has differential, but mild effects.

Voss K, Gamblin TC - Mol Neurodegener (2009)

ARA induction of tau isoform polymerization. 2 μM tau isoforms were incubated with various ARA concentrations (0 μM to 100 μM) for 18 hrs. Polymerization was measured by (A) LLS or (B) ThS fluorescence. Symbols correspond as follows: (open square) 0N3R, (closed square) 0N4R, (open triangle) 1N3R, (closed triangle) 1N4R, (open circle) 2N3R, (closed circle) 2N4R, and (closed diamond) assembly incompetent protein (AIP (I277, 308P)). Data is in arbitrary units and represents an average of 3 trials ± SEM. After 18 hours, ARA induced polymerization reactions were visualized by TEM at 20,000x magnification. Images are as follows: (C) 0N3R, (D) 1N3R, (E) 2N3R, (F) 0N4R, (G) 1N4R, and (H) 2N4R. Each isoform image is representative of polymerized material at 75 μM ARA. Scale bars represent 1 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683827&req=5

Figure 2: ARA induction of tau isoform polymerization. 2 μM tau isoforms were incubated with various ARA concentrations (0 μM to 100 μM) for 18 hrs. Polymerization was measured by (A) LLS or (B) ThS fluorescence. Symbols correspond as follows: (open square) 0N3R, (closed square) 0N4R, (open triangle) 1N3R, (closed triangle) 1N4R, (open circle) 2N3R, (closed circle) 2N4R, and (closed diamond) assembly incompetent protein (AIP (I277, 308P)). Data is in arbitrary units and represents an average of 3 trials ± SEM. After 18 hours, ARA induced polymerization reactions were visualized by TEM at 20,000x magnification. Images are as follows: (C) 0N3R, (D) 1N3R, (E) 2N3R, (F) 0N4R, (G) 1N4R, and (H) 2N4R. Each isoform image is representative of polymerized material at 75 μM ARA. Scale bars represent 1 μm.
Mentions: Because the primary sequences of the tau isoforms are different, the various combinations of exons could influence their interactions with inducer molecules or cause conformational changes affecting tau-tau interactions during the polymerization process. The polymerization characteristics of each isoform was determined by incubating 2 μM protein with a range of ARA concentrations (0–150 μM) and monitoring the extent of polymerization with right angle laser light scattering (LLS, Figure 2a) and thioflavine S fluorescence (ThS, Figure 2b). We determined that above 100 μM ARA, the results were highly variable and not easily reproducible (data not shown). This is consistent with previously published results showing high variability at the inhibitory ratio of inducer to 2N4R tau [7]. The isoforms followed the same general trend of increasing amounts of polymerization with increasing inducer concentrations. 4R tau isoforms (containing exon 10) polymerized to a greater extent than 3R isoforms (Figure 2a & 2b), indicating that the additional microtubule binding repeat enhances tau polymerization.

Bottom Line: We have found that each isoform has a unique microtubule binding and polymerization profile that is altered by GSK-3beta.These results indicate that tau phosphorylation by a single kinase can have isoform specific outcomes.The mild nature of these changes, however, makes it unlikely that differential effects of GSK-3beta phosphorylation on the isoforms are causative in neurodegenerative disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA. gamblin@ku.edu.

ABSTRACT

Background: Tau protein exists as six different isoforms that differ by the inclusion or exclusion of exons 2, 3 and 10. Exon 10 encodes a microtubule binding repeat, thereby resulting in three isoforms with three microtubule binding repeats (3R) and three isoforms that have four microtubule binding repeats (4R). In normal adult brain, the relative amounts of 3R tau and 4R tau are approximately equal. These relative protein levels are preserved in Alzheimer's disease, although in other neurodegenerative tauopathies such as progressive supranuclear palsy, corticobasal degeneration and Pick's disease, the ratio of 3R:4R is frequently altered. Because tau isoforms are not equally involved in these diseases, it is possible that they either have inherently unique characteristics owing to their primary structures or that post-translational modification, such as phosphorylation, differentially affects their properties.

Results: We have determined the effects of phosphorylation by a kinase widely believed to be involved in neurodegenerative processes, glycogen synthase kinase-3beta (GSK-3beta), on the microtubule binding and inducer-initiated polymerization of these isoforms in vitro. We have found that each isoform has a unique microtubule binding and polymerization profile that is altered by GSK-3beta. GSK-3beta phosphorylation had differential effects on the isoforms although there were similarities between isoforms and the effects were generally mild.

Conclusion: These results indicate that tau phosphorylation by a single kinase can have isoform specific outcomes. The mild nature of these changes, however, makes it unlikely that differential effects of GSK-3beta phosphorylation on the isoforms are causative in neurodegenerative disease. Instead, the inherent differences in the isoform interactions themselves and local conditions in the diseased cells are likely the major determinant of isoform involvement in various neurodegenerative disorders.

No MeSH data available.


Related in: MedlinePlus