Microtubule-associated protein tau is known to be hyperphosphorylated in Alzheimer disease brain and this abnormal hyperphosphorylation is associated with an inability of tau to promote the assembly of microtubule in the affected neurons. Our previous studies demonstrated that abnormally phosphorylated tau could be dephosphorylated after treatment with alkaline phosphatase, thereby suggesting that the abnormal phosphorylation of tau might in part be the result of a deficiency of the phosphoprotein phosphatase system in patients with Alzheimer disease. In the present study we used 32P-labeled phosphorylase kinase and poly(Glu, Tyr) 4:1 as substrates to measure phosphoprotein phosphatase activities in Alzheimer disease and control brains. The activities of phosphoseryl/phosphothreonyl-protein phosphatase types 1, 2A, 2B, and 2C and of phosphotyrosyl-protein phosphatase in frontal gray and white matters from 13 Alzheimer brains were determined and compared with those from 12 age-matched control brains. The activities of type 1 phosphatase and phosphotyrosyl phosphatase in gray matter and of type 2A phosphatase in both gray and white matters were significantly lower in Alzheimer disease brains than in controls. These findings suggest that the hyperphosphorylation of tau in Alzheimer disease brain could result from a protein dephosphorylation defect in vivo. The decrease in the phosphatase activities in Alzheimer disease might also be involved in the formation of beta-amyloid by augmenting the amyloidogenic pathway processing of beta-amyloid precursor protein.
PHF-tau, which is pbosphorylated at 10 SerfFhr-Pro and 11 non-SerfFhr-Pro sites, is unable to promote microtubule assembly. Phosphorylation of the non-Ser/Thr-Pro site, Ser-262, is reported to be primarily responsible for this. The identifies of kinnse(s) responsible for Ser-262 phnspborylation are still to be clarified. In this study we have used the monoclonal antibody 12E8, which recognizes P-Set-262 and P-Set-356 on tau, to survey different kinases for their abilities to phospborylate Ser-262 on human tau 3L (tau410). In decreasing order of effectiveness we found that Ser-262 and Ser-356 phosphorylation can be catalyzed by CaM kinase II >> C-kinase >> GSK-3-= Akinase>>CK-l. CaM kinase II and C-kinase were shown to phosphorylate both Ser-262 and Ser-356. The binding of tau to taxol-stabilized microtubules was decreased by 35 and 42% after phosphorylation by CaM kinase II and C-kinase, respectively. Of the fraction of tun that bound to microtubules, about 50% was phosphorylated at Ser-262 and Ser-356. These results suggest that Ser-262 and Ser-356 are very good substrates for CaM kinase II but their phosphorylations are not sufficient to achieve maximal inhibition of tau binding to microtubules.
Tau protein from Alzheimer disease (AD) brain is phosphorylated at eleven Ser/Thr-Pro and nine Serfrhr-X sites. The former sites are phosphorylated by proline-dependent protein kinases (PDPKs), the latter by non-PDPKs. The identities of both the PDPKs and non-PDPKs involved in AD tau hyperphosphorylation are still to be established. In this study we have analyzed the interactions between a PDPK (GSK-3) and several non-PDPKs (A-kinase, C-kinase, CK-1, CaM kinase II) in the phosphorylation of one isoform (tau 39) of human tau. We found that the rate of phosphorylation of tan 39 by GSK-3 was increased several-fold if tau were first prephosphorylated by the non-PDPKs. Further, several Alzbeimer-like epitopes in tau can be induced only slowly after phosphorylation of tau by GSK-3 alone. After a prephosphorylation of tau by the non-PDPKs, however, the rate of induction of these epitopes by GSK-3 is increased several-fold. These results suggest that one role of non-PDPK-catalyzed phosphorylation is the modulation of PDPK-catalyzed phosphorylation of tau in AD brain.
The phosphorylation of bovine tau, either by GSK-3 alone or by a combination of GSK3 and several non-prolinedependent protein kinases (non-PDPKs), was studied. GSK3 alone catalyzed the incorporation of -3 mol 32P/mol tau at a relatively slow rate. Prephosphorylation of tau by A-kinase, Ckinase, or CK-2 (but not by CK-1, CaM kinase II or Gr kinase) increased both the rate and extent of a subsequent phosphorylation catalyzed by GSK3 by several-fold. These results suggest that the phosphorylation of tau by PDPKs such as GSK3 (and possibly MAP kinase, cdk5) may be positively modulated at the substrate level by non-PDPK-catalyzed phosphorylations.
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