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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

Heparin induction of tau isoform polymerization. 2 μM tau isoforms were mixed with various heparin concentrations (0 mg/ml to 0.024 mg/ml) and incubated 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. Data is in arbitrary units and represents an average of 3 trials ± SEM except for 2N4R with 0.006 and 0.018 mg/ml heparin, 1N4R with 0.024 mg/ml heparin, and 0N4R with 0.018 mg/ml heparin which were 2 trials. After 18 hours, heparin induced polymerization reactions were visualized by TEM at 20,000× 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 0.006 mg/ml heparin. Scale bars represent 1 μm.
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Figure 3: Heparin induction of tau isoform polymerization. 2 μM tau isoforms were mixed with various heparin concentrations (0 mg/ml to 0.024 mg/ml) and incubated 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. Data is in arbitrary units and represents an average of 3 trials ± SEM except for 2N4R with 0.006 and 0.018 mg/ml heparin, 1N4R with 0.024 mg/ml heparin, and 0N4R with 0.018 mg/ml heparin which were 2 trials. After 18 hours, heparin induced polymerization reactions were visualized by TEM at 20,000× 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 0.006 mg/ml heparin. Scale bars represent 1 μm.

Mentions: Because it is known that some isoforms do not polymerize equally in the presence of heparin [21], the polymerization characteristics for all six isoforms were determined under conditions of low ionic strength [22,23]. The isoforms (2 μM) were incubated in the presence of varying concentrations (0–0.024 mg/ml) of heparin and polymerization was measured by LLS (Figure 3a) and ThS fluorescence (Figure 3b). Polymerization was detected by both methods for the 4R isoforms, and the dose-response curve was biphasic, indicating an optimal inducer concentration for polymerization. 2N4R required a lower concentration of heparin for optimal polymerization than 0N4R and 1N4R. Under these heparin induction conditions, the 3R isoforms failed to polymerize to detectable levels above background (Figure 3a and 3b). 4R isoforms (Figure 3f–h) were induced to form long filaments, but 3R isoforms (Figure 3c–e) did not polymerize into any observable filaments by TEM.


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

Voss K, Gamblin TC - Mol Neurodegener (2009)

Heparin induction of tau isoform polymerization. 2 μM tau isoforms were mixed with various heparin concentrations (0 mg/ml to 0.024 mg/ml) and incubated 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. Data is in arbitrary units and represents an average of 3 trials ± SEM except for 2N4R with 0.006 and 0.018 mg/ml heparin, 1N4R with 0.024 mg/ml heparin, and 0N4R with 0.018 mg/ml heparin which were 2 trials. After 18 hours, heparin induced polymerization reactions were visualized by TEM at 20,000× 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 0.006 mg/ml heparin. Scale bars represent 1 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Heparin induction of tau isoform polymerization. 2 μM tau isoforms were mixed with various heparin concentrations (0 mg/ml to 0.024 mg/ml) and incubated 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. Data is in arbitrary units and represents an average of 3 trials ± SEM except for 2N4R with 0.006 and 0.018 mg/ml heparin, 1N4R with 0.024 mg/ml heparin, and 0N4R with 0.018 mg/ml heparin which were 2 trials. After 18 hours, heparin induced polymerization reactions were visualized by TEM at 20,000× 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 0.006 mg/ml heparin. Scale bars represent 1 μm.
Mentions: Because it is known that some isoforms do not polymerize equally in the presence of heparin [21], the polymerization characteristics for all six isoforms were determined under conditions of low ionic strength [22,23]. The isoforms (2 μM) were incubated in the presence of varying concentrations (0–0.024 mg/ml) of heparin and polymerization was measured by LLS (Figure 3a) and ThS fluorescence (Figure 3b). Polymerization was detected by both methods for the 4R isoforms, and the dose-response curve was biphasic, indicating an optimal inducer concentration for polymerization. 2N4R required a lower concentration of heparin for optimal polymerization than 0N4R and 1N4R. Under these heparin induction conditions, the 3R isoforms failed to polymerize to detectable levels above background (Figure 3a and 3b). 4R isoforms (Figure 3f–h) were induced to form long filaments, but 3R isoforms (Figure 3c–e) did not polymerize into any observable filaments by TEM.

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