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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

Changes in microtubule binding of tau isoforms when phosphorylated with GSK-3β. Microtubule binding reactions were performed at varying concentrations of tau (0 μM to 10 μM) and bound vs. free tau (in μM) for both unphosphorylated (black circle) and phosphorylated (red circle) was plotted (A-F). Three separate trials are plotted together to demonstrate the variability in the reactions. The data were fit to a nonlinear single site binding equation for unphosphorylated (black solid line) and phosphorylated (red solid line) tau isoforms (A-F). The 95% confidence intervals of the fits are drawn as dashed lines. Outliers existed for phosphorylated samples, are shown as red open boxes, and were excluded from calculations. (G) The dissociation constant (Kd) (in μM) was determined for unphosphorylated (white bars) and phosphorylated (red bars) isoforms by Kd = the concentration of tau required to reach 1/2 Bmax. Data represents the average of 3 trials ± SEM.
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Figure 5: Changes in microtubule binding of tau isoforms when phosphorylated with GSK-3β. Microtubule binding reactions were performed at varying concentrations of tau (0 μM to 10 μM) and bound vs. free tau (in μM) for both unphosphorylated (black circle) and phosphorylated (red circle) was plotted (A-F). Three separate trials are plotted together to demonstrate the variability in the reactions. The data were fit to a nonlinear single site binding equation for unphosphorylated (black solid line) and phosphorylated (red solid line) tau isoforms (A-F). The 95% confidence intervals of the fits are drawn as dashed lines. Outliers existed for phosphorylated samples, are shown as red open boxes, and were excluded from calculations. (G) The dissociation constant (Kd) (in μM) was determined for unphosphorylated (white bars) and phosphorylated (red bars) isoforms by Kd = the concentration of tau required to reach 1/2 Bmax. Data represents the average of 3 trials ± SEM.

Mentions: Tau is a microtubule-associated protein that, when phosphorylated at specific sites in the microtubule binding regions, such as at site S262 by p110MARK (microtubule affinity regulating kinase), promotes strong disassociation (~10×) from microtubules [25], while phosphorylation outside of the microtubule binding regions (such as by GSK-3β in the proline rich region) has weaker effects on microtubule binding [26]. We therefore determined the effects of phosphorylation by GSK-3β on the microtubule binding affinities of each isoform, using unphosphorylated protein as a control (Figure 5a–f). Unphosphorylated 4R isoforms bound more tightly than the unphosphorylated 3R isoforms (Figure 5g), which is consistent with previously published results [27]. Upon phosphorylation, the largest decrease in microtubule affinities occurred in 1N isoforms (~3×) (Figure 5g), indicating GSK-3β phosphorylates 1N isoforms at sites that mildly influence microtubule binding. The average Kd of phosphorylated 0N3R, 0N4R, and 2N4R isoforms was higher than the corresponding unphosphorylated Kd, however, these changes did not reach statistically significant levels (Figure 5g). 2N3R showed an increase in affinity for microtubules upon GSK-3β phosphorylation. These data indicate that phosphorylation by GSK-3β has mild effects on the microtubule binding of tau isoforms, which is consistent with phosphorylation at the proline rich region or other regions only mildly involved with microtubule binding.


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

Voss K, Gamblin TC - Mol Neurodegener (2009)

Changes in microtubule binding of tau isoforms when phosphorylated with GSK-3β. Microtubule binding reactions were performed at varying concentrations of tau (0 μM to 10 μM) and bound vs. free tau (in μM) for both unphosphorylated (black circle) and phosphorylated (red circle) was plotted (A-F). Three separate trials are plotted together to demonstrate the variability in the reactions. The data were fit to a nonlinear single site binding equation for unphosphorylated (black solid line) and phosphorylated (red solid line) tau isoforms (A-F). The 95% confidence intervals of the fits are drawn as dashed lines. Outliers existed for phosphorylated samples, are shown as red open boxes, and were excluded from calculations. (G) The dissociation constant (Kd) (in μM) was determined for unphosphorylated (white bars) and phosphorylated (red bars) isoforms by Kd = the concentration of tau required to reach 1/2 Bmax. Data represents the average of 3 trials ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Changes in microtubule binding of tau isoforms when phosphorylated with GSK-3β. Microtubule binding reactions were performed at varying concentrations of tau (0 μM to 10 μM) and bound vs. free tau (in μM) for both unphosphorylated (black circle) and phosphorylated (red circle) was plotted (A-F). Three separate trials are plotted together to demonstrate the variability in the reactions. The data were fit to a nonlinear single site binding equation for unphosphorylated (black solid line) and phosphorylated (red solid line) tau isoforms (A-F). The 95% confidence intervals of the fits are drawn as dashed lines. Outliers existed for phosphorylated samples, are shown as red open boxes, and were excluded from calculations. (G) The dissociation constant (Kd) (in μM) was determined for unphosphorylated (white bars) and phosphorylated (red bars) isoforms by Kd = the concentration of tau required to reach 1/2 Bmax. Data represents the average of 3 trials ± SEM.
Mentions: Tau is a microtubule-associated protein that, when phosphorylated at specific sites in the microtubule binding regions, such as at site S262 by p110MARK (microtubule affinity regulating kinase), promotes strong disassociation (~10×) from microtubules [25], while phosphorylation outside of the microtubule binding regions (such as by GSK-3β in the proline rich region) has weaker effects on microtubule binding [26]. We therefore determined the effects of phosphorylation by GSK-3β on the microtubule binding affinities of each isoform, using unphosphorylated protein as a control (Figure 5a–f). Unphosphorylated 4R isoforms bound more tightly than the unphosphorylated 3R isoforms (Figure 5g), which is consistent with previously published results [27]. Upon phosphorylation, the largest decrease in microtubule affinities occurred in 1N isoforms (~3×) (Figure 5g), indicating GSK-3β phosphorylates 1N isoforms at sites that mildly influence microtubule binding. The average Kd of phosphorylated 0N3R, 0N4R, and 2N4R isoforms was higher than the corresponding unphosphorylated Kd, however, these changes did not reach statistically significant levels (Figure 5g). 2N3R showed an increase in affinity for microtubules upon GSK-3β phosphorylation. These data indicate that phosphorylation by GSK-3β has mild effects on the microtubule binding of tau isoforms, which is consistent with phosphorylation at the proline rich region or other regions only mildly involved with microtubule binding.

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