Novel regulation of death‐associated protein kinase 1 in Alzheimer’s disease
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Abstract Background Death‐associated protein kinase 1 (DAPK1) is a serine/threonine kinase that plays an important role in regulating neuronal function and is genetically linked to Alzheimer’s disease (AD). We previously showed that DAPK1 expression is markedly increased in 75% of human AD brains. Moreover, we found that DAPK1 promotes tau protein accumulation and its phosphorylation at multiple AD‐related sites. However, it is not known whether and how DAPK1 expression or activity is regulated in AD, including tau hyperphosphorylation and functions. Method We investigated the effects of melatonin on DAPK1 regulation by examining DAPK1 protein stability, and tau hyperphosphorylation and functions using comprehensive approaches in cell culture models, ex vivo mouse models, and human patient tissues. We synthesized biotin‐melatonin to demonstrate direct binding of melatonin and DAPK1. Moreover, we assessed the correlation of melatonin‐regulated DAPK1 expression and Pin1 phosphorylation because DAPK1 increases tau hyperphosphorylation through inhibiting the catalytic activity of Pin1. Result We discovered that melatonin critically regulates DAPK1 by reducing its protein stability, thereby attenuating tau hyperphosphorylation in AD. Melatonin decreases DAPK1 expression in a dose‐ and time‐dependent manner in primary neurons and neuronal cell lines. Moreover, the regulation of DAPK1 by melatonin is not dependent on DAPK1 transcription because DAPK1 mRNA levels are not affected by melatonin. Melatonin promotes the ubiquitination of DAPK1 and decreases its protein stability through a proteasome‐dependent pathway. Furthermore, melatonin directly interacts with the ankyrin repeat domain of DAPK1. Inhibition of DAPK1 by melatonin and a pharmacological DAPK1 inhibitor synergistically reduces tau expression and phosphorylation at multiple AD‐related sites. Melatonin and a DAPK1 inhibitor promote neurite outgrowth and microtubule assembly, which are disrupted by tau hyperphosphorylation. Mechanistically, melatonin‐mediated DAPK1 degradation increases the activity of Pin1, a prolyl isomerase known to play a protective role against tau hyperphosphorylation and AD. Finally, DAPK1 and melatonin levels are inversely correlated in the brains of human AD patients. Conclusion Our study demonstrates a novel role of melatonin in the direct regulation of DAPK1, and that synergistic targeting of DAPK1 by melatonin and DAPK1 inhibitors offers an attractive approach to block tau hyperphosphorylation in AD.Keywords:
Hyperphosphorylation
Tau protein
Microtubules (MTs), primarily composed of α and β tubulin polymers, must often work in concert with microtubule-associated proteins (MAPs) in order to modulate their functional demands. In a mature brain neuron, one of the key MAPs that resides primarily in the axonal compartment is the tau protein. Tau, in the adult human brain, is a set of six protein isoforms, whose binding affinity to MTs can be modulated by phosphorylation. In addition to the role that phosphorylation of tau plays in the “normal” physiology of neurons, hyperphosphorylated tau is the primary component of the fibrillary pathology in Alzheimer's disease (AD). Although many protein kinases are known to phosphorylate tau in vitro, the in vivo players contributing to the hyperphosphorylation of tau remain elusive. The experiments in this study attempt to define which protein kinases and protein phosphatases reside in the associated network of microtubules, thereby being strategically positioned to influence the phosphorylation of tau. Microtubule fractions are utilized to determine which of the microtubule-associated kinases most readily impacts the phosphorylation of tau at “AD-like” sites. Results from this study indicate that PKA, CK1, GSK3β, and cdk5 associate with microtubules. Among the MT-associated kinases, GSK3β and cdk5 most readily contribute to the ATP-induced “AD-like” phosphorylation of tau. J. Neurosci. Res. 62:463–472, 2000. © 2000 Wiley-Liss, Inc.
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Cyclin-dependent kinase 5
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Alzheimer disease (AD) and related tauopathies are all characterized histopathologically by neurofibrillary degeneration. The neurofibrillary changes, whether of paired helical filaments (PHF), twisted ribbons or straight filaments (SF) are made up of abnormally hyperphosphorylated tau. Unlike normal tau which promotes assembly and maintains structure of microtubules, the abnormal tau not only lacks these functions but also sequesters normal tau, MAP1 and MAP2, and causes disassembly of microtubules. This toxic behavior of the abnormal tau is solely due to its hyperphosphorylation because dephosphorylation restores it into a normal-like protein. The abnormal hyperphosphorylation also promotes the self-assembly of tau into PHF/SF. The state of phosphorylation of a phosphoprotein is the function of the activities of protein kinases and as well as of protein phosphatases that regulate the level of phosphorylation. A cause of the abnormal hyperphosphorylation in AD brain is a decrease in the activity of protein phosphatase (PP)-2A, a major regulator of the phosphorylation of tau. A decrease in PP-2A activity results in the abnormal hyperphosphorylation of tau not only by decreased dephosphorylation of tau but also by stimulating the activities of tau kinases like CaMKII, PKA and MAP kinases which are regulated by PP-2A. Thus, the abnormal hyperphosphorylation can be inhibited both by inhibition of the activity/s of a tau protein kinase and as well as by restoration of the activity/s of a tau protein phosphatase. The development of drugs that inhibit neurofibrillary degeneration is a very promising and feasible therapeutic approach to inhibit the progression of AD and related tauopathies.
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Tau is a microtubule‐associated protein that is abnormally hyperphosphorylated in the filamentous lesions that define a number of neurodegenerative diseases collectively referred to as tauopathies. We previously showed that stress‐activated protein (SAP) kinases phosphorylate tau protein at many of the hyperphosphorylated sites in vitro. Here we have developed a system to study the effects of five SAP kinases (SAPK1c/JNK1, SAPK2a/p38α, SAPK2b/p38β, SAPK3/p38γ and SAPK4/p38δ) on tau phosphorylation in intact cells. All kinases phosphorylated tau, albeit at different efficiencies. Tau was a good substrate for SAPK3/p38γ and SAPK4/p38δ, a reasonable substrate for SAPK2b/p38β and a relatively poor substrate for SAPK2a/p38α and SAPK1c/JNK1. These findings indicate that the aberrant activation of SAP kinases, especially SAPK3/p38γ and SAPK4/p38δ, could play an important role in the abnormal hyperphosphorylation of tau that is an invariant feature of the tauopathies.
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Abstract Tau is a neuronal microtubule‐associated protein. Its hyperphosphorylation plays a critical role in Alzheimer disease (AD). Expression and phosphorylation of tau are regulated developmentally, but its dynamic regulation and the responsible kinases or phosphatases remain elusive. Here, we studied the developmental regulation of tau in rats during development from embryonic day 15 through the age of 24 months. We found that tau expression increased sharply during the embryonic stage and then became relatively stable, whereas tau phosphorylation was much higher in developing brain than in mature brain. However, the extent of tau phosphorylation at seven of the 14 sites studied was much less in developing brain than in AD brain. Tau phosphorylation during development matched the period of active neurite outgrowth in general. Tau phosphorylation at various sites had different topographic distributions. Several tau kinases appeared to regulate tau phosphorylation collectively at overlapping sites, and the decrease of overall tau phosphorylation in adult brain might be due to the higher levels of tau phosphatases in mature brain. These studies provide new insight into the developmental regulation of site‐specific tau phosphorylation and identify the likely sites required for the abnormal hyperphosphorylation of tau in AD.
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Tau protein from Alzheimer disease (AD) brain is phosphorylated at eleven Ser/Thr‐Pro and nine Ser/Thr‐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 tau 39 by GSK‐3 was increased several‐fold if tau were first prephosphorylated by the non‐PDPKs. Further, several Alzheimer‐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.
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Hyperphosphorylation of microtubule-associated protein tau is thought to contribute to Alzheimer's disease (AD) pathogenesis. We previously showed that DNA damage-activated cell cycle checkpoint kinases Chk1 and Chk2 phosphorylate tau at an AD-related site and enhance tau toxicity, suggesting potential roles of these kinases in AD. The purpose of this study is to systematically identify which sites in tau are directly phosphorylated by Chk1 and Chk2. Using recombinant human tau phosphorylated by Chk1 and Chk2 in vitro, we first analyzed tau phosphorylation at the AD-related sites by Western blot with phospho-tau-specific antibodies. Second, to globally identify phosphorylated sites in tau, liquid chromatography–tandem mass spectrometry (LC–MS3) was employed. These systematic analyses identified a total of 27 Ser/Thr residues as Chk1- or Chk2- target sites. None of them were proline-directed kinase targets. Many of these sites are located within the microtubule-binding domain and C-terminal domain, whose phosphorylation has been shown to reduce tau binding to microtubules and/or has been implicated in tau toxicity. Among these 27 sites, 13 sites have been identified to be phosphorylated in AD brains. Since DNA damage is accumulated in diseased brains, Chk1 and Chk2 may be involved in tau phosphorylation and toxicity in AD pathogenesis.
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Checkpoint Kinase 2
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Neurofibrillary tangles and neuropil threads, the main forms of tau lesions in AD, exhibit a characteristic temporal and spatial distribution pattern that strongly indicates a direct causal connection to the disease progression. In spite of intense research, however, the primary impetus that initiates formation of these insoluble lesions remains elusive. It is generally assumed that hyperphosphorylation of tau is an essential component of the pathological tau cascade and that it is brought about by an imbalance between kinases and phosphatases. In our work we tested the possibility that altered conformation of the substrate (truncated tau) influences its phosphorylation status. We used a rat model of tauopathy expressing a human truncated tau as a transgene. By western blotting we compared the phosphorylation levels of endogenous rat tau, truncated transgenic tau and the levels of the relevant tau kinases. The transgene, truncated tau in a rat model of tauopathies, exhibited high phosphorylation levels throughout the life of the animals, while the normal endogenous rat tau proteins were unaffected. Furthermore, transgenic animals did not exhibit elevated levels of most relevant tau kinases or their activated phospho-forms when compared with wild type animals. In vitro analysis of the phosphorylation rate of the recombinant human tau proteins in an in vitro kinase reaction revealed that truncated human tau becomes phosphorylated faster and to a higher degree than the full length tau protein at several phosphorylation sites relevant for AD pathology. We present evidence that conformational abnormalities of the truncated tau protein are sufficient to induce its abnormal phosphorylation and initiate the cascade of events leading to the formation of the insoluble misfolded forms.
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Abstract Microtubule‐associated protein tau in a hyperphosphorylated state is the major component of the filamentous lesions that define a number of neurodegenerative diseases commonly referred to as tauopathies. Hyperphosphorylation of tau at most sites appears to precede filament assembly. Many of the hyperphosphorylated sites are serine/threonine–proline sequences. Here we show that c‐Jun N‐terminal kinases JNK1, JNK2 and JNK3 phosphorylate tau at many serine/threonine–prolines, as assessed by the generation of the epitopes of phosphorylation‐dependent anti‐tau antibodies. Of the three protein kinases, JNK2 phosphorylated the most sites in tau, followed by JNK3 and JNK1. Phosphorylation by JNK isoforms resulted in a greatly reduced ability of tau to promote microtubule assembly. These findings extend the number of candidate protein kinases for the hyperphosphorylation of tau in Alzheimer's disease and other neurodegenerative disorders.
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