Effect of Pin1 or Microtubule Binding on Dephosphorylation of FTDP-17 Mutant Tau

Abstract

Neurodegenerative tauopathies, including Alzheimer disease, are characterized by abnormal hyperphosphorylation of the microtubule-associated protein Tau. One group of tauopathies, known as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), is directly associated with mutations of the gene tau. However, it is unknown why mutant Tau is highly phosphorylated in the patient brain. In contrast to in vivo high phosphorylation, FTDP-17 Tau is phosphorylated less than wild-type Tau in vitro. Because phosphorylation is a balance between kinase and phosphatase activities, we investigated dephosphorylation of mutant Tau proteins, P301L and R406W. Tau phosphorylated by Cdk5-p25 was dephosphorylated by protein phosphatases in rat brain extracts. Compared with wildtype Tau, R406W was dephosphorylated faster and P301L slower. The two-dimensional phosphopeptide map analysis suggested that faster dephosphorylation of R406W was due to a lack of phosphorylation at Ser-404, which is relatively resistant to dephosphorylation. We studied the effect of the peptidylprolyl isomerase Pin1 or microtubule binding on dephosphorylation of wild-type Tau, P301L, and R406W in vitro. Pin1 catalyzes the cis/trans isomerization of phospho-Ser/Thr-Pro sequences in a subset of proteins. Dephosphorylation of wildtype Tau was reduced in brain extracts of Pin1-knockout mice, and this reduction was not observed with P301L and R406W. On the other hand, binding to microtubules almost abolished dephosphorylation of wild-type and mutant Tau proteins. These results demonstrate that mutation of Tau and its association with microtubules may change the conformation of Tau, thereby suppressing dephosphorylation and potentially contributing to the etiology of tauopathies.

Isomerase Pin1 Stimulates Dephosphorylation of Tau Protein at Cyclin-dependent Kinase (Cdk5)-dependent Alzheimer Phosphorylation Sites

“…Tau-We have reported previously that recombinant Tau phosphorylated by Cdk5-p35 is dephosphorylated by PP2A faster in WT mouse brain extract than in Pin1-deficient mouse brain extract (39). This result suggests that Pin1 recognizes Cdk5-phosphorylated Tau to stimulate its dephosphorylation.…”

“…These Pin1 mutants did not bind to phosphorylated Tau (Fig. 2C) (8,11,29,39,50). Some of these sites may be minor sites that were identified with phosphorylation-specific antibodies.…”

“…Hence, Pin1 could be a critical regulator of Tau dephosphorylation to (i) restore physiological Tau function such as microtubule binding and (ii) suppress neurofibrillary tangle formation by enhancing dephosphorylation by PP2A. We reported recently that Pin1 stimulates dephosphorylation of Tau phosphorylated by Cdk5-p25, suggesting that there are more Pin1 binding motifs in Tau (39). The Pin1 binding sites in Tau were shown to be phosphoThr-231 (34,40) and phospho-Thr-212 (41).…”

Bioinformatic Prediction and Confirmation of β-Adducin as a Novel Substrate of Glycogen Synthase Kinase 3

“…␤-Adducin is the third substrate of Cdk5 demonstrated to display relative resistance to phosphatases (others being CRMP2 and Tau (42,43)), but this is the first time a GSK3 phosphosite has been demonstrated to display relative resistance. We previously showed that resistance to phosphatases was in part due to the presence of a basic residue at the ϩ3 position, which stabilizes the negative charge on the phosphorylated serine (42).…”

“…This suggests that other kinases might also target these sites or that other kinases can compensate for the loss of Cdk5 and GSK3 activity. Alternatively, removal of phosphate from these sites by phosphatases might be very inefficient, as has previously been shown for phosphosites in CRMP2 and Tau that are relatively resistant to dephosphorylation by phosphatases (42,43). To determine whether any phosphosites in ␤-adducin display similar resistance to phosphatases, a rat brain lysate was incubated without phosphatase inhibitors at 30°C for up to 4 h. Relative rates of dephosphorylation at each site by endogenous phosphatases were measured using Western blotting.…”