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To whom correspondence may be addressed: Dept. of Neurochemistry, New York State Institute for Basic Research, 1050 Forest Hill Rd., Staten Island, NY 10314. Tel.: 718-494-5390; Fax: 718-494-1080;
To whom correspondence may be addressed: Dept. of Neurochemistry, New York State Institute for Basic Research, 1050 Forest Hill Rd., Staten Island, NY 10314. Tel.: 718-494-5248; Fax: 718-494-1080;
Affiliations
New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314
* This work was supported by the New York State Office of Mental Retardation and Developmental Disabilities; Alzheimer Association Grant NIRG-03-4721 (to F. L.); National Institutes of Health Grants AG16760 (to C.-X. G.), AG19158 (to K. I.), and NS31221 (to S. R.); and an award from the Li Foundation, Inc. USA (to C.-X. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Protein phosphatase (PP) 5 is highly expressed in the mammalian brain, but few physiological substrates have yet been identified. Here, we investigated the kinetics of dephosphoryation of phospho-tau by PP5 and found that PP5 had a Km of 8–13 μm toward tau, which is similar to that of PP2A, the major known tau phosphatase. This Km value is within the range of intraneuronal tau concentration in human brain, suggesting that tau could be a physiological substrate of both PP5 and PP2A. PP5 dephosphorylated tau at all 12 Alzheimer's disease (AD)-associated abnormal phosphorylation sites studied, with different efficiency toward each site. Thr205, Thr212, and Ser409 of tau were the most favorable sites; Ser199, Ser202, Ser214, Ser396, and Ser404 were less favorable sites; and Ser262 was the poorest site for PP5. Overexpression of PP5 in PC12 cells resulted in dephosphorylation of tau at multiple phosphorylation sites. The activity but not the protein level of PP5 was found to be decreased by ∼20% in AD neocortex. These results suggest that tau is probably a physiological substrate of PP5 and that the abnormal hyperphosphorylation of tau in AD might result in part from the decreased PP5 activity in the diseased brains.
Tau protein is a major microtubule-associated protein in neurons. The known biological function of tau protein is to stimulate and stabilize microtubule formation from tubulin subunits. In human brain, there are six tau isoforms generated by alternative mRNA splicing from a single gene (
). Tau is a phosphoprotein that normally contains 2–3 phosphates/molecule, but it is abnormally hyperphosphorylated with a stoichiometry of 9–10 moles of phosphate per mole of protein in Alzheimer's disease (AD)
). To date, more than 30 phosphorylation sites have been identified in AD hyperphosphorylated tau, some of which are not phosphorylated in normal tau (for review, see Refs.
). However, the molecular mechanism by which tau becomes abnormally hyperphosphorylated in AD brain is not yet well understood. Theoretically, the abnormal hyperphosphorylation of tau could be the result of up-regulation of tau kinases or down-regulation of tau phosphatases. Several studies have shown that three major protein phosphatases (PPs), PP1, PP2A, and PP2B, dephosphorylate tau in vitro (
). Nevertheless, PP2A does not dephosphorylate all the phosphorylation sites of tau, and down-regulation of PP2A in animal brain could not produce PHFs (
). These studies suggest that in addition to PP2A, other phosphatases or factors might also participate in the regulation of tau phosphorylation.
PP5 is a 58-KDa phosphoseryl/phosphothreonyl protein phosphatase. It is ubiquitously expressed in all types of mammalian tissue, with a relatively high level in the brain (
). PP5 contains a C-terminal catalytic domain structurally related to the PP1/PP2A/PP2B family and an N-terminal regulatory domain consisting of three tetratricopeptide repeats (TPRs) that usually mediate protein-protein interaction. It has been demonstrated that PP5 interacts with multiple target proteins through the TPR domain (for review, see Ref.
). In the absence of activators, PP5 exhibits unusually low phosphatase activity in vitro. However, PP5, after removal of its TPR domain or in the presence of long-chain fatty acyl-CoA ester or long-chain fatty acids, effectively dephosphorylates p-nitrophenyl phosphate (pNPP) as well as phosphoproteins such as casein, myelin basic protein, reduced carboxamidomethylated and maleylated lysozyme, and tau (
) that PP5 dephosphorylates tau in vitro and may associate with microtubules, raising the possibility that PP5 may also participate in regulation of tau phosphorylation in the brain. To elucidate the role of PP5 in regulation of tau phosphorylation and in the hyperphosphorylation of tau in AD, we studied the kinetics of tau dephosphorylation by PP5, mapped the sites of tau dephosphorylated by PP5, investigated tau dephosphorylation by PP5 in cultured cells, and measured PP5 activity in AD and control brains. Our results suggest that PP5 may play a role in regulation of tau phosphorylation and that a reduction of PP5 activity may contribute to hyperphosphorylation of tau in AD.
EXPERIMENTAL PROCEDURES
Materials—Recombinant rat PP5, cyclin-dependent kinase 5 and its activator, p25, and the largest isoform of human tau, tau441, were cloned, expressed, and purified as described previously (
). The catalytic subunit of cAMP-dependent kinase was purchased from Sigma. All phosphorylation-dependent and site-specific tau antibodies were from Biosource (Table I). Peroxidase-conjugated anti-mouse and anti-rabbit IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA). 125I-labeled anti-mouse and anti-rabbit IgG and ECL kit were from Amersham Biosciences. The phosphorylation-independent tau antibodies 92e and R134d and anti-PP5 antibody were raised in rabbits as described previously (
Brain Tissue—Medial temporal gyrus of six AD brains (one male and five females; age, 86.5 ± 6.5 years; post-mortem delay, 2.6 ± 0.5 h) and seven age-matched normal human brains (two males and five females; age, 86.6 ± 2.9 years; post-mortem delay, 2.9 ± 0.4 h) used for this study were obtained from the Sun Health Research Institute Donation Program (Sun City, AZ). All brain samples were pathologically confirmed and stored at –70 °C until used.
Preparation of Phosphorylated Tau (P-tau) and 32P-labeled Tau— Recombinant tau441 was phosphorylated with cAMP-dependent protein kinase and cyclin-dependent kinase 5/p25 by incubating tau441 (2 mg/ml) with the catalytic subunit of cAMP-dependent protein kinase (10 μg/ml) in buffer consisting of 40 mm HEPES, pH 6.8, 10 mm β-mercaptoethanol, 10 mm MgCl2, 1.0 mm EGTA, and 0.2 mm ATP at 30 °C for 90 min. The pH of the reaction mixture was then adjusted to 7.4 with 1 N NaOH, followed by addition of cyclin-dependent kinase 5/p25 (60 μg/ml) and additional 0.2 mm ATP. After incubation at 30 °C for another 4 h, the reaction mixture was desalted by using Sephadex G-50. Fractions containing P-tau were pooled and stored at –20 °C. When used for radioactive phosphatase assays, nonradioactive ATP was substituted with [γ-32P]ATP (750 × 106 μCi/mol) for preparation of 32P-tau. Under these conditions, ∼3 moles of phosphate were added to each mole of tau441.
PP5 Activity Assays—Unless specified, PP5 assays were performed in a reaction mixture (15 μl) containing 50 mm Tris-HCl, pH 7.4, 10 mm β-mercaptoethanol, 5 μm arachidonyl-CoA (AA-CoA), and 0.1 mg/ml 32P-tau. After incubation at 30 °C for 20 min, the reaction was terminated, and the released 32Pi was determined by Cerenkov counting after separation from 32P-tau by ascending paper chromatography as described previously (
For the kinetic analyses, PP5 was assayed by using increasing concentrations (0.5–40 μm) of 32P-tau, and the Km value of PP5 was calculated by using the Lineweaver-Burk double-reciprocal method. The catalytic efficiency (Kcat/Km) was calculated by dividing the turnover number (Kcat, the number of molecules of substrate turned into product by 1 molecule of enzyme in 1 s) by the Km value.
Dephosphorylation of P-tau by PP5—P-tau (0.2 mg/ml) was incubated with 0.5 μg/ml PP5 at 30 °C for 2 h in a reaction mixture containing 50 mm Tris-HCl, pH 7.4, 10 mm β-mercaptoethanol, and 5 μm AA-CoA. The reaction was stopped by adding 0.33 volume of 4-fold concentrated sample buffer for SDS-PAGE. After heating in boiling water for 5 min, the samples were subjected to SDS-PAGE and Western blot analysis using site-specific and phosphorylation-dependent tau antibodies as described previously (
For quantitative detection of site-specific dephosphorylation of tau by PP5, the dephosphorylation reaction was carried out by using the same condition as described above for PP5 activity assay, except that P-tau was used to replace 32P-tau. The reaction was stopped by addition of a mixture of phosphatase inhibitors (50 mm sodium phosphate, pH 7.5, 5 μm okadaic acid, 50 mm NaF, 5 mm EDTA, and 5 mm EGTA). The dephosphorylation of P-tau at each specific site was measured by using a dot-blot radioimmunoassay as described previously (
Immunoprecipitation of PP5—Rat brain or autopsy human brain tissue was homogenized with 9 volumes of buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 10 mm β-mercaptoethanol, and 2 μg/ml each of aprotinin, leupeptin, and pepstatin. The 16,000 × g extracts were prepared, and the protein concentrations were measured by the Bradford method (
). The diluted extracts were then incubated with anti-PP5 antibody that was pre-bound to protein G-agarose beads (Pierce) for 4 h at 4 °C. The negative control was prepared with protein G-agarose beads without anti-PP5. The immunoprecipitated complex was washed three times with Tris-buffered saline and two times with Tris-HCl buffer and used for Western blots or PP5 activity assay.
Transfection of PC12 Cells and PP5—pCI plasmids encoding full-length rat PP5 (pCI-FLAG-PP5499) or C-terminal truncated, lipid-independent active PP5 (pCI-FLAG-PP5486) were constructed as described previously (
), except that the parent vector was modified to include an N-terminal FLAG epitope tag and a short linker sequence (LGGGATR) immediately preceding the coding sequence for PP5. PC12 cells with stable expression of tau441, which were cloned as described previously (
), were transiently transfected with either pCI-FLAG-PP5499 or pCI-FLAG-PP5486 by using Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer's instructions. Forty-eight hours later, the cells were harvested and lysed in SDS-PAGE sample buffer, followed by Western blot analysis as described previously (
). We therefore studied the activation of PP5 using AA-CoA, the most abundant intracellular long-chain fatty acyl-CoA, by using recombinant tau phosphorylated by cAMP-dependent protein kinase and cyclin-dependent kinase 5/p25 as a substrate. We found that AA-CoA caused a concentration-dependent stimulation of PP5 activity toward P-tau. Stimulation was seen with a concentration of AA-CoA as low as 0.3 μm and reached a plateau at ∼5 μm AA-CoA (Fig. 1A). At 20 μm AA-CoA, PP5 activity toward P-tau was stimulated by 25-fold. The AA-CoA concentration required for half-maximal activation (AC50) was 1.2 μm (Fig. 1A, inset). When pNPP, a commonly used non-protein substrate for phosphatases, was used as substrate, AA-CoA stimulated PP5 activity with a slightly lower AC50 and maximal stimulation (Fig. 1A).
Fig. 1Kinetics of PP5-catalyzed dephosphorylation of tau.A, stimulation of PP5 activity by AA-CoA. PP5 activity was assayed by using 0.1 mg/ml 32P-tau (•) or 50 mm pNPP (○) as substrate in the presence of various concentrations of AA-CoA, as indicated. The data are presented as stimulation (fold) of PP5 activity over basal activity assayed in the absence of AA-CoA. Inset, expanded scale of data at the lowest concentrations of AA-CoA used to calculate the AC50 of each curve. B, kinetic analysis of tau dephosphorylation by PP5. The velocity of dephosphorylation of 32P-tau was measured at various concentrations of P-tau in the presence 5 μm AA-CoA and 0.1 μg/ml PP5. C, data shown in B were plotted using the Lineweaver-Burk double-reciprocal method. The x-axis intercept was used to estimate Km.
We then measured the Km value of PP5-catalyzed dephosphorylation of P-tau in the presence of 5 μm AA-CoA. We observed typical Michaelis-Menten kinetics of dephosphorylation of P-tau by PP5 (Fig. 1B). Plot of the data by the Lineweaver-Burk double-reciprocal method demonstrated a very good fit (Fig. 1C). The Km calculated from the Lineweaver-Burk equation was 7.6 μm. The calculated catalytic efficiency (Kcat/Km) was 98 × 103 s–1m–1. As a reference, we also measured the Km of PP5-catalyzed dephosphorylation of pNPP and found a Km of 64 mm, which is in agreement with those reported by Ramsey and Chinkers (
), our results suggest that tau is probably a physiological substrate of PP5.
The above-mentioned kinetic studies were carried out by using recombinant rat PP5 expressed from Escherichia coli.To learn whether recombinant PP5 catalyzes dephosphorylation of tau with the same kinetics as brain PP5, we compared the Km of recombinant PP5 with that of PP5 immuno-affinity-purified from rat brain and human brain extracts. Western blot analyses demonstrated that anti-PP5 successfully immunoprecipitated most of PP5 from brain extract but did not co-precipitate PP1, PP2A, or PP2B, the three major protein phosphatases in mammalian brain (Fig. 2A). Binding of our anti-PP5 to PP5 did not block its phosphatase activity, as demonstrated by dose-dependent phosphatase activity of the immunoprecipitates (Fig. 2B). This is consistent with the fact that the PP5 antiserum recognizes the linker region between the TPR and catalytic domains rather than catalytic domain of PP5 (
). We then studied the kinetics of brain PP5 toward P-tau and found that the Km values of rat brain PP5 and human brain PP5 were similar to that of recombinant rat PP5 (Fig. 2C and Table II). Therefore, we used recombinant PP5 for the rest of this study. The Vmax of brain PP5 calculated from Fig. 2C was not comparable with that of the recombinant PP5 calculated from Fig. 1C because the exact amount of brain PP5 used for these assays was not known.
Fig. 2Immunoprecipitation of rat brain PP5 and dephosphorylation of P-tau.A, PP5 was immunoprecipitated with anti-PP5 pre-coupled to protein G-agarose. The immunocomplex was analyzed with Western blots developed with antibodies against PP1, PP2A, PP2B, and PP5. Arrowheads indicate IgG (anti-PP5) that was recognized by anti-rabbit secondary antibodies. B, dephosphorylation of 32P-tau by increasing amounts of PP5 immunocomplex isolated from rat brain (bottom panel). The top panel shows a Western blot of the PP5 immunocomplex used for this assay. C, kinetic analysis of tau dephosphorylation by rat brain PP5. The velocity of dephosphorylation of 32P-tau by rat brain PP5 was measured at various concentrations of 32P-tau in the presence 5 μm of AA-CoA (top panel). The bottom panel is the Lineweaver-Burk plot of these data. The x-axis intercept was used to calculate the Km of rat brain PP5 toward P-tau.
PP5 Dephosphorylates P-tau at Multiple Sites—Phosphorylation of different sites within tau differentially impacts the biological activity of tau and the polymerization of tau into PHFs. To map to the exact sites that are dephosphorylated by PP5, we used a number of phosphorylation-dependent tau antibodies that recognize tau phosphorylation at individual sites. As shown in Fig. 3, a combination of cAMP-dependent protein kinase and cyclin-dependent kinase 5 phosphorylated recombinant tau at Thr181, Ser199, Ser202, Thr205, Thr212, Ser214, Thr231, Ser235, Ser262, Ser396, Ser404, and Ser409. An up-shift of the gel mobility of tau was also seen after phosphorylation (Fig. 3, A and B, compare lanes 2 with lanes 1). Treatment of P-tau with PP5 abolished the staining of all these phosphorylation-dependent antibodies and changed the gel mobility to the same as that for unphosphorylated tau (Fig. 3, compare lanes 3 with lanes 2). These results indicated that PP5 dephosphorylates tau at all the above-mentioned phosphorylation sites studied.
Fig. 3Dephosphorylation of tau at multiple sites. P-tau after incubation at 30 °C for 2 h in dephosphorylation buffer in the presence (lanes 3) or absence (lanes 2) of 0.1 μg/ml PP5 was analyzed by Western blots using site-specific and phosphorylation-dependent tau antibodies as indicated below each panel (A–N). Lanes 1, as a control, contained the same amount of tau before phosphorylation. Coomassie Blue staining (A) and antibody R134d (B), which recognizes both phosphorylated and non-phosphorylated tau, were included to show total tau and the gel mobility shift.
We further investigated the efficiency of tau dephosphorylation by PP5 at individual phosphorylation sites. The site-specific dephosphorylation of tau was measured by a dot-blot radioimmunoassay developed with the corresponding site-specific tau antibodies. Comparison of the extent of dephosphorylation at specific sites as a function of PP5 concentration indicated that PP5 dephosphorylated tau most efficiently at Thr205, Thr212, and Ser409; moderately at Ser199, Ser202, Ser214, Ser396, and Ser404; and least efficiently at Ser262 (Fig. 4). Similar results were also obtained when the time courses of the dephosphorylation at various sites were studied (data not shown). Because the signal for Thr181, Thr231, and Ser235 of P-tau was low compared with those of other site-specific antibodies (see Fig. 3), the quantitative dephosphorylation of tau at these sites was not measured.
Fig. 4Quantitation of tau dephosphorylation by PP5 at individual phosphorylation sites. P-tau was incubated with various concentrations of PP5 for 20 min, and the reactions were measured by a dot-blot radioimmunoassay developed with antibodies that only react with tau phosphorylated at the indicated individual sites.
PP5 Dephosphorylates Tau in Living Cells—We also investigated whether PP5 dephosphorylates tau in living cells by using PC12 cells that stably expressed human tau441 (
). We found that after transfection for 48 h, the expression of recombinant PP5 was ∼5-fold higher than that of endogenous PP5 as estimated by Western blots developed with anti-PP5 (Fig. 5A). The expression level of active PP5486 was similar to or slightly lower than that of full-length PP5499 as estimated according to the staining intensity with anti-PP5. When several tau antibodies, each of which only recognizes tau phosphorylated at its specific phosphorylation site, were used to study the effect of PP5 overexpression on tau phosphorylation, we found that the transient transfection of either PP5499 or PP5486 in PC12 cells resulted in decreased staining of tau with these phosphorylation-dependent tau antibodies, indicating dephosphorylation of tau at all the phosphorylation sites studied (Fig. 5, B–H). At several phosphorylation sites (Ser199, Ser202, and Ser214), tau was dephosphorylated to a larger extent when transfected with the lipid-independent, active PP5486 than with full-length PP5499. Because these two forms of PP5 were expressed at comparable levels, this result is consistent with the conclusion that PP5486, which is more active than PP5499in vitro, promotes more extensive dephosphorylation of tau in living cells. The identical amounts of tau loaded into each lane of these blots were confirmed by using phosphorylation-independent tau antibody R134d and an anti-FLAG antibody (the recombinant tau441 contained a FLAG tag) (Fig. 5, I and J). These results suggested that PP5 regulated phosphorylation of tau in the cell.
Fig. 5Dephosphorylation of tau by PP5 in PC12 cells. Tau441-expressing PC12 cells were transfected with empty plasmids as a control (Mock), plasmids of full-length PP5 (pCI-FLAG-PP5499, PP5499), or plasmids of lipid-independent active PP5 (pCI-FLAG-PP5486, PP5486) for 48 h. Then, the cell lysates (5.0 μg/lane) were analyzed by Western blots developed with anti-PP5 to determine PP5 expression levels (A), phosphorylation-dependent and site-specific tau antibodies to determine tau phosphorylation levels at the specific phosphorylation sites (B–H), and R134d and anti-FLAG to determine the levels of tau441 (I and J). Note that due to a FLAG tag of recombinant PP5, full-length PP5499 had a slightly larger apparent molecular weight than endogenous PP5, whereas truncated active PP5486 migrated to the same position as endogenous PP5 (A). Unlike endogenous PP5, the transfected PP5, for a reason unknown at present, showed as two bands.
PP5 Activity Is Decreased in AD Brain—To investigate whether there is a dysregulation of PP5 in AD brain that could theoretically contribute to abnormal hyperphosphorylation of tau, we measured PP5 activity of extracts from AD and age-matched control brains by using P-tau as a substrate. We found an ∼20% reduction of PP5 activity in AD brains as compared with controls (Fig. 6A). To learn whether the reduction of PP5 activity was due to a reduction of the phosphatase protein level, we measured the level of PP5 in the extracts by using quantitative Western blots and found no change in PP5 level in AD brains as compared with controls (Fig. 6B). These results indicate that the activity, but not the level, of PP5 was down-regulated in AD brain.
Fig. 6Activity and level of PP5 in AD and control brains.A, activities of PP5 immunoprecipitated from equivalent amounts of extracts from temporal cortices of six AD and seven control brains were assayed using 32P-tau as a substrate. Means ± S.E. are presented. *, p < 0.05 by Student's t test as compared with control group. B, PP5 protein levels of the extracts were analyzed by Western blots developed with anti-PP5 (top panel). The blot was quantitated by densitometry, and the data are presented as mean ± S.E. (bottom panel).
We showed previously that PP5 dephosphorylates tau protein and that it is localized in the neuronal cytoplasm in human brain and appears to associate with microtubules (
). These studies suggest that tau might be a physiological substrate of PP5. In the present study, we investigated the kinetics of tau dephosphorylation by PP5 and found that PP5 had a Km of 8–13 μm and a catalytic efficiency (Kcat/Km) of 98 × 103 s–1m–1. This Km value is similar to that of PP2A, a known tau phosphatase, toward tau (
), we estimate that there is 5–10 μm tau in neurons. The Km value of PP5 toward tau is at the range of the intraneuronal tau concentration. These data are consistent with the possibility that tau is a physiological substrate of PP5 and that PP5 participates in the regulation of tau phosphorylation.
PP5 has little phosphatase activity in vitro in the absence of added lipid or without removal of the TPR domain or C terminus by limited proteolysis (
). Early work showed that unsaturated long-chain fatty acids such as arachidonic acid increased PP5 activity toward both pNPP and artificial protein substrates (
) recently found that physiological concentrations of long-chain fatty acyl-CoA derivatives dramatically activate PP5 toward pNPP. However, this is not true when myelin basic protein is used as a substrate (
), indicating that, as has been seen for other members of this PP family, activation maybe substrate-dependent. In the present study, we found that even 0.625 μm AA-CoA stimulated PP5 activity toward tau and that the activation was maximal at 5–10 μm AA-CoA. This stimulation profile is similar to that seen when pNPP is used as a substrate (
). Thus, PP5 might be active toward tau in vivo in the presence of the physiologic levels of fatty acyl-CoA derivatives. Our results showing dephosphorylation of tau in PC12 cells after overexpression of full-length PP5499 demonstrated that PP5 can indeed act on tau in living cells.
Phosphorylation of tau at different sites differentially impacts the biological activity of tau and the polymerization of tau into PHFs (
). In the present studies, we used recombinant tau441 as a substrate after phosphorylation with cAMP-dependent protein kinase and cyclin-dependent kinase 5, a non-proline-directed protein kinase and a proline-directed protein kinase, respectively, which are among the most physiologically relevant tau kinases (for review, see Ref.
). With a combination of these two kinases, tau was phosphorylated at least at 12 phosphorylation sites (Fig. 3), all of which are hyperphosphorylated in AD brain. Hence, this in vitro phosphorylated tau is an ideal tau substrate for studying tau phosphatases. Quantitative analyses of PP5-catalyzed dephoshorylation of tau at each individual site indicated that PP5 dephosphorylates tau most efficiently at Thr205, Thr212, and Ser409 but poorly at Ser262. In contrast, phospho-Ser262 of tau is a favorable substrate for PP2A,
). These studies suggest that tau phosphorylation can be regulated by more than one phosphatase and that each phosphatase may regulate tau phosphorylation at different sites with certain preference.
Many studies have demonstrated that abnormal hyperphosphorylation of tau is important in the pathogenesis of AD (for review, see Ref.
) have reported previously that the activity of PP2A is decreased in selected areas of AD brain, which suggested that the abnormal hyperphosphorylation of tau might result from the pathological reduction of PP2A activity in AD brain. However, in cultured cells, metabolically active rat brain slices, and in vivo, inhibition of PP2A induces hyperphosphorylation of tau at some, but not all, of the abnormally hyperphosphorylated sites seen in AD and fails to produce neurofibrillary tangles in the brain (
). Hence, the reduction of PP2A activity may only partially account for the abnormal tau hyperphosphorylation in AD brain. In this study, we found that PP5 activity was also decreased in AD brain. Because PP5 and PP2A appear to target differential phosphorylation sites of tau, reduction in the activities of both PP5 and PP2A may contribute to the abnormal hyperphosphorylation of tau in AD brain. It will be interesting to elucidate the relative contributions of PP5 and PP2A to the regulation of tau phosphorylation at each phosphorylation site.
Despite the decreased PP5 activity in AD brain, the protein level of PP5 was found to be the same in AD brain as that of controls. The mechanism leading to a decrease in PP5 activity in AD brain is currently not known. Although free arachidonic acid and its phospholipid forms were found to be decreased in selected regions of AD brain, these decreases do not contribute to the decreased PP5 activity we observed because the immunoprecipitated PP5 was used for the activity assay and the assays were carried out in the presence of 5 μm AA-CoA. One possible mechanism leading to the decreased PP5 activity in AD brain might be some posttranslational or structural modification that may down-regulate PP5 catalytic activity. Another possibility is the presence of a PP5 inhibitor in AD brain that might co-immunoprecipitate with PP5. Because the catalytic domain of PP5 is similar to that of PP2A (
), it is possible that the same inhibitor might underlie the reduction of both PP5 and PP2A activities in AD brain. The mechanism by which PP5 activity is reduced in AD brain requires further investigation.
In summary, we have studied the kinetics of PP5-catalyzed tau dephosphorylation, mapped the tau phosphorylation sites targeted by PP5, investigated the dephosphorylation of tau by PP5 in PC12 cells, and measured PP5 activity in AD and control human brains. We found that (a) the Km value of PP5 toward tau is similar to that of PP2A and is within the range of intracellular tau concentration of human brain, (b) PP5 dephosphorylates tau at least at 12 phosphorylation sites with different efficiencies, (c) PP5 can dephosphorylate tau in living cells, and (d) PP5 activity, but not protein level, is decreased in AD brain. These results suggest that tau is a physiological substrate of PP5 and that the abnormal hyperphosphorylation of tau in AD brain might result in part from the decreased activity of PP5.
Acknowledgments
We thank Drs. T. Beach and L. I. Sue of Sun Health Research Institute Donation Program for brain tissue, Dr. E. El-Akkad for the preparation of recombinant tau, and Dr. B.-K. Hahm for expression and purification of PP5.
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