Pinning down phosphorylated tau and tauopathies

Neurofibrillary tangles (NFTs) are prominent neuronal lesions in a large subset of neurodegenerative diseases, including Alzheimer's disease (AD). NFTs are mainly composed of insoluble Tau that is hyperphosphorylated on many serine or threonine residues preceding proline (pSer/Thr-Pro). Tau hyperphosphorylation abolishes its biological function to bind microtubules and promotes microtubule assembly and precedes neurodegeneration. Not much is known about how tau is further regulated following phosphorylation. Notably, we have recently shown that phosphorylated Ser/Thr-Pro motifs exist in two distinct conformations. The conversion between two conformations in some proteins is catalyzed by the prolyl isomerase Pin1. Pin1 binds to tau phosphorylated specifically on the Thr231-Pro site and probably catalyzes cis/trans isomerization of pSer/Thr-Pro motif(s), thereby inducing conformational changes in tau. Such conformational changes can directly restore the ability of phosphorylated Tau to bind microtubules and promote microtubule assembly and/or facilitate tau dephosphorylation by its phosphatase PP2A, as PP2A activity is conformation-specific. Furthermore, Pin1 expression inversely correlates with the predicted neuronal vulnerability in normally aged brain and also with actual neurofibrillary degeneration in AD brain. Moreover, deletion of the gene encoding Pin1 in mice causes progressive age-dependent neuropathy characterized by motor and behavioral deficits, tau hyperphosphorylation, tau filament formation and neuronal degeneration. Distinct from all other mouse models where transgenic overexpression of specific proteins elicits tau-related pathologies, Pin1 is the first protein whose depletion causes age-dependent neurodegeneration and tau pathologies. Thus, Pin1 is pivotal in maintaining normal neuronal function and preventing age-dependent neurodegeneration. This could represent a promising interventive target to prevent neurodegenerative diseases.     

Pin1-dependent prolyl isomerization regulates dephosphorylation of Cdc25C and tau proteins

The reversible protein phosphorylation on serine or threonine residues that precede proline (pSer/Thr-Pro) is a key signaling mechanism for the control of various cellular processes, including cell division. The pSer/Thr-Pro moiety in peptides exists in the two completely distinct cis and trans conformations whose conversion is catalyzed specifically by the essential prolyl isomerase Pin1. Previous results suggest that Pin1 might regulate the conformation and dephosphorylation of its substrates. However, it is not known whether phosphorylation-dependent prolyl isomerization occurs in a native protein and/or affects dephosphorylation of pSer/Thr-Pro motifs. Here we show that the major Pro-directed phosphatase PP2A is conformation-specific and effectively dephosphorylates only the trans pSer/Thr-Pro isomer. Furthermore, Pin1 catalyzes prolyl isomerization of specific pSer/Thr-Pro motifs both in Cdc25C and tau to facilitate their dephosphorylation by PP2A. Moreover, Pin1 and PP2A show reciprocal genetic interactions, and prolyl isomerase activity of Pin1 is essential for cell division in vivo. Thus, phosphorylation-specific prolyl isomerization catalyzed by Pin1 is a novel mechanism essential for regulating dephosphorylation of certain pSer/Thr-Pro motifs.     

Enzyme-linked enzyme-binding assay for Pin1 WW domain ligands

Peptidyl prolyl cis-trans isomerase (PPIase) interacting with NIMA-1 (Pin1) catalyzes the cis-trans isomerization of pSer/pThr-Pro amide bonds. Pin1 is a two-domain protein that represents a promising target for the treatment of cancer. Both domains of Pin1 bind the pSer/pThr-Pro motif; PPIase enzymatic activity occurs in the catalytic domain, and the WW domain acts as a recognition module for the pSer/pThr-Pro motif. An assay we call an enzyme-linked enzyme-binding assay (ELEBA) was developed to measure the K_d of ligands that bind selectively to the WW domain. A ligand specific for the WW domain of Pin1 was covalently immobilized in a 96-well plate. Commercially available Pin1 conjugated to horseradish peroxidase was used for chemiluminescent detection of ligands that block the association of the WW domain with immobilized ligand. The peptide ligands were derived from the cell cycle regulatory phosphatase, Cdc25c, residues 45-50. The K_d values for Fmoc-VPRpTPVGGGK-NH₂ and Ac-VPRpTPV-NH₂ were determined to be 36 ± 4 and 110 ± 30 μM, respectively. The ELEBA offers a selective approach for detecting ligands that bind to the Pin1 WW domain, even in the presence of the catalytic domain. This method may be applied to any dual specificity, multidomain protein.     

Phosphorylation-specific prolyl isomerization: is there an underlying theme?

The prolyl isomerase Pin1 is a conserved enzyme that is intimately involved in diverse biological processes and pathological conditions such as cancer and Alzheimer's disease. By catalysing cis-trans interconversion of certain motifs containing phosphorylated serine or threonine residues followed by a proline residue (pSer/Thr-Pro), Pin1 can have profound effects on phosphorylation signalling. The structural and functional differences that result from cis-trans isomerization of specific pSer/Thr-Pro motifs probably underlie most, if not all, Pin1-dependent actions. Phosphorylation-dependent prolyl isomerization by Pin1 remains a unique mode for the modulation of signal transduction. Here, we provide an overview of the plethora of regulatory events that involve this unique enzyme, with a particular focus on oncogenic signalling and neurodegeneration.     

Pin1 modulates the dephosphorylation of the RNA polymerase II C-terminal domain by yeast Fcp1

The reversible phosphorylation of serine and threonine residues N-terminal to proline (pSer/Thr-Pro) is an important signaling mechanism in the cell. The pSer/Thr-Pro moiety exists in the two distinct cis and trans conformations, whose conversion is catalyzed by the peptidyl-prolyl isomerase (PPIase) Pin1. Among others, Pin1 binds to the phosphorylated C-terminal domain (CTD) of the largest subunit of the RNA polymerase II, but the biochemical and functional relevance of this interaction is unknown. Here we confirm that the CTD phosphatase Fcp1 can suppress a Pin1 mutation in yeast. Furthermore, this genetic interaction requires the phosphatase domain as well as the BRCT domain of Fcp1, suggesting a critical role of the Fcp1 localization. Based on these observations, we developed a new in vitro assay to analyze the CTD dephosphorylation by Fcp1 that uses only recombinant proteins and mimics the in vivo situation. This assay allows us to present strong evidence that Pin1 is able to stimulate CTD dephosphorylation by Fcp1 in vitro, and that this stimulation depends on Pin1's PPIase activity. Finally, Pin1 significantly increased the dephosphorylation of the CTD on the Ser(5)-Pro motif, but not on Ser(2)-Pro in yeast, which can be explained with Pin1's substrate specificity. Together, our results indicate a new role for Pin1 in the regulation of CTD phosphorylation and present a further example for prolyl isomerization-dependent protein dephosphorylation.     

Molecular mechanisms of the phospho-dependent prolyl cis/trans isomerase Pin1

Since its discovery 10 years ago, Pin1, a prolyl cis/trans isomerase essential for cell cycle progression, has been implicated in a large number of molecular processes related to human diseases, including cancer and Alzheimer's disease. Pin1 is made up of a WW interaction domain and a C-terminal catalytic subunit, and several high-resolution structures are available that have helped define its function. The enzymatic activity of Pin1 towards short peptides containing the pSer/Thr-Pro motif has been well documented, and we discuss the available evidence for the molecular mechanisms of its isomerase activity. We further focus on those studies that examine its cis/trans isomerase function using full-length protein substrates. The interpretation of this research has been further complicated by the observation that many of its pSer/Thr-Pro substrate motifs are located in natively unstructured regions of polypeptides, and are characterized by minor populations of the cis conformer. Finally, we review the data on the possibility of alternative modes of substrate binding and the complex role that Pin1 plays in the degradation of its substrates. After considering the available work, it seems that further analysis is required to determine whether binding or catalysis is the primary mechanism through which Pin1 affects cell cycle progression.     

Pin1 in Alzheimer's disease: multiple substrates, one regulatory mechanism?

Presence of neuritic plaques and neurofibrillary tangles in the brain are two neuropathological hallmarks of Alzheimer's disease (AD), although the molecular basis of their coexistence remains elusive. The neurofibrillary tangles are composed of microtubule binding protein Tau, whereas neuritic plaques consist of amyloid-beta peptides derived from amyloid precursor protein (APP). Recently, the peptidyl-prolyl cis/trans isomerase Pin1 has been identified to regulate the function of certain proteins after phosphorylation and to play an important role in cell cycle regulation and cancer development. New data indicate that Pin1 also regulates the function and processing of Tau and APP, respectively, and is important for protecting against age-dependent neurodegeneration. Furthermore, Pin1 is the only gene known so far that, when deleted in mice, can cause both Tau and Abeta-related pathologies in an age-dependent manner, resembling many aspects of human Alzheimer's disease. Moreover, in the human AD brain Pin1 is downregulated or inhibited by oxidative modifications and/or genetic changes. These results suggest that Pin1 deregulation may provide a link between formation of tangles and plaques in AD.     

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

Neurodegenerative diseases associated with the pathological aggregation of microtubule-associated protein Tau are classified as tauopathies. Alzheimer disease, the most common tauopathy, is characterized by neurofibrillary tangles that are mainly composed of abnormally phosphorylated Tau. Similar hyperphosphorylated Tau lesions are found in patients with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) that is induced by mutations within the tau gene. To further understand the etiology of tauopathies, it will be important to elucidate the mechanism underlying Tau hyperphosphorylation. Tau phosphorylation occurs mainly at proline-directed Ser/Thr sites, which are targeted by protein kinases such as GSK3β and Cdk5. We reported previously that dephosphorylation of Tau at Cdk5-mediated sites was enhanced by Pin1, a peptidyl-prolyl isomerase that stimulates dephosphorylation at proline-directed sites by protein phosphatase 2A. Pin1 deficiency is suggested to cause Tau hyperphosphorylation in Alzheimer disease. Up to the present, Pin1 binding was only shown for two Tau phosphorylation sites (Thr-212 and Thr-231) despite the presence of many more hyperphosphorylated sites. Here, we analyzed the interaction of Pin1 with Tau phosphorylated by Cdk5-p25 using a GST pulldown assay and Biacore approach. We found that Pin1 binds and stimulates dephosphorylation of Tau at all Cdk5-mediated sites (Ser-202, Thr-205, Ser-235, and Ser-404). Furthermore, FTDP-17 mutant Tau (P301L or R406W) showed slightly weaker Pin1 binding than non-mutated Tau, suggesting that FTDP-17 mutations induce hyperphosphorylation by reducing the interaction between Pin1 and Tau. Together, these results indicate that Pin1 is generally involved in the regulation of Tau hyperphosphorylation and hence the etiology of tauopathies.     

Relative resistance of Cdk5-phosphorylated CRMP2 to dephosphorylation

Collapsin response mediator protein 2 (CRMP2) binds to microtubules and regulates axon outgrowth in neurons. This action is regulated by sequential phosphorylation by the kinases cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 (GSK3) at sites that are hyperphosphorylated in Alzheimer disease. The increased phosphorylation in Alzheimer disease could be due to increases in Cdk5 and/or GSK3 activity or, alternatively, through decreased activity of a CRMP phosphatase. Here we establish that dephosphorylation of CRMP2 at the residues targeted by GSK3 (Ser-518/Thr-514/Thr-509) is carried out by a protein phosphatase 1 family member in vitro, in neuroblastoma cells, and primary cortical neurons. Inhibition of GSK3 activity using insulin-like growth factor-1 or the highly selective inhibitor CT99021 causes rapid dephosphorylation of CRMP2 at these sites. In contrast, pharmacological inhibition of Cdk5 using purvalanol results in only a gradual and incomplete dephosphorylation of CRMP2 at the site targeted by Cdk5 (Ser-522), suggesting a distinct phosphatase targets this residue. A direct comparison of dephosphorylation at the Cdk5 versus GSK3 sites in vitro shows that the Cdk5 site is comparatively resistant to phosphatase treatment. The presence of the peptidyl-prolyl isomerase enzyme, Pin1, does not affect dephosphorylation of Ser-522 in vitro, in cells, or in Pin1 transgenic mice. Instead, the relatively high resistance of this site to phosphatase treatment is at least in part due to the presence of basic residues located nearby. Similar sequences in Tau are also highly resistant to phosphatase treatment. We propose that relative resistance to phosphatases might be a common feature of Cdk5 substrates and could contribute to the hyperphosphorylation of CRMP2 and Tau observed in Alzheimer disease.     

Regulation of Pin1 peptidyl-prolyl cis/trans isomerase activity by its WW binding module on a multi-phosphorylated peptide of Tau protein

The WW module of the peptidyl-prolyl cis/trans isomerase Pin1 targets specifically phosphorylated proteins involved in the cell cycle through the recognition of phospho-Thr(Ser)-Pro motifs. When the microtubule-associated Tau protein becomes hyperphosphorylated, it equally becomes a substrate for Pin1, with two recognition sites described around the phosphorylated Thr212 and Thr231. The Pin1 WW domain binds both sites with moderate affinity, but only the Thr212-Pro213 bond is isomerized by the catalytic domain of Pin1. We show here that, in a peptide carrying a single recognition site, the WW module increases significantly the enzymatic isomerase activity of Pin1. However, with addition of a second recognition motif, the affinity of both the WW and catalytic domain for the substrate increases, but the isomerization efficacy decreases. We therefore conclude that the WW domain can act as a negative regulator of enzymatic activity when multiple phosphorylation is present, thereby suggesting a subtle mechanism of its functional regulation.     

Death-associated protein kinase 1 phosphorylates Pin1 and inhibits its prolyl isomerase activity and cellular function

Pin1 is a phospho-specific prolyl isomerase that regulates numerous key signaling molecules and whose deregulation contributes to disease notably cancer. However, since prolyl isomerases are often believed to be constitutively active, little is known whether and how Pin1 catalytic activity is regulated. Here, we identify death-associated protein kinase 1 (DAPK1), a known tumor suppressor, as a kinase responsible for phosphorylation of Pin1 on Ser71 in the catalytic active site. Such phosphorylation fully inactivates Pin1 catalytic activity and inhibits its nuclear location. Moreover, DAPK1 inhibits the ability of Pin1 to induce centrosome amplification and cell transformation. Finally, Pin1 pSer71 levels are positively correlated with DAPK1 levels and negatively with centrosome amplification in human breast cancer. Thus, phosphorylation of Pin1 Ser71 by DAPK1 inhibits its catalytic activity and cellular function, providing strong evidence for an essential role of the Pin1 enzymatic activity for its cellular function.     

Prolyl isomerase Pin1 promotes amyloid precursor protein (APP) turnover by inhibiting glycogen synthase kinase-3β (GSK3β) activity: novel mechanism for Pin1 to protect against Alzheimer disease

Alzheimer disease (AD) is characterized by the presence of senile plaques of amyloid-β (Aβ) peptides derived from amyloid precursor protein (APP) and neurofibrillary tangles made of hyperphosphorylated Tau. Increasing APP gene dosage or expression has been shown to cause familial early-onset AD. However, whether and how protein stability of APP is regulated is unclear. The prolyl isomerase Pin1 and glycogen synthase kinase-3β (GSK3β) have been shown to have the opposite effects on APP processing and Tau hyperphosphorylation, relevant to the pathogenesis of AD. However, nothing is known about their relationship. In this study, we found that Pin1 binds to the pT330-P motif in GSK3β to inhibit its kinase activity. Furthermore, Pin1 promotes protein turnover of APP by inhibiting GSK3β activity. A point mutation either at Thr-330, the Pin1-binding site in GSK3β, or at Thr-668, the GSK3β phosphorylation site in APP, abolished the regulation of GSK3β activity, Thr-668 phosphorylation, and APP stability by Pin1, resulting in reduced non-amyloidogenic APP processing and increased APP levels. These results uncover a novel role of Pin1 in inhibiting GSK3β kinase activity to reduce APP protein levels, providing a previously unrecognized mechanism by which Pin1 protects against Alzheimer disease.     

The peptidylprolyl cis/trans-isomerase Pin1 modulates stress-induced dephosphorylation of Tau in neurons. Implication in a pathological mechanism related to Alzheimer disease

Deregulation of Tau phosphorylation is a key question in Alzheimer disease pathogenesis. Recently, Pin1, a peptidylprolyl cis/trans-isomerase, was proposed to be a new modulator in Tau phosphorylation in Alzheimer disease. In vitro, Pin1 was reported to present a high affinity for both Thr(P)-231, a crucial site for microtubule binding, and Thr(P)-212. In fact, Pin1 may facilitate Thr(P)-231 dephosphorylation by protein phosphatase 2A through trans isomerization of the Thr(P)-Pro peptide bound. However, whether Pin1 binding to Tau leads to isomerization of a single site or of multiple Ser/Thr(P)-Pro sites in vivo is still unknown. In the present study, Pin1 involvement was investigated in stress-induced Tau dephosphorylation with protein phosphatase 2A activation. Both oxidative (H2O2) and heat stresses induced hypophosphorylation of a large set of phospho-Tau epitopes in primary cortical cultures. In both cases, juglone, a Pin1 pharmacological inhibitor, partially prevented dephosphorylation of Tau at Thr-231 among a set of phosphoepitopes tested. Moreover, Pin1 is physiologically found in neurons and partially co-localized with Tau. Furthermore, in Pin1-deficient neuronal primary cultures, H2O2 stress-induced Tau dephosphorylation at Thr(P)-231 was significantly lower than in wild type neurons. Finally, Pin1 transfection in Pin1-deficient neuronal cell cultures allowed for rescuing the effect of H2O2 stress-induced Tau dephosphorylation, whereas a Pin1 catalytic mutant did not. This is the first demonstration of an in situ Pin1 involvement in a differential Tau dephosphorylation on the full-length multiphosphorylated substrate.     

Peptide binding induces large scale changes in inter-domain mobility in human Pin1

Pin1 is a peptidyl-prolyl cis/trans isomerase (PPIase) essential for cell cycle regulation. Pin1-catalyzed peptidyl-prolyl isomerization provides a key conformational switch to activate phosphorylation sites with the common phospho-Ser/Thr-Pro sequence motif. This motif is ubiquitously exploited in cellular response to a variety of signals. Pin1 is able to bind phospho-Ser/Thr-Pro-containing sequences at two different sites that compete for the same substrate. One binding site is located within the N-terminal WW domain, which is essential for protein targeting and localization. The other binding site is located in the C-terminal catalytic domain, which is structural homologous to the FK506-binding protein (FKBP) class of PPIases. A flexible linker of 12 residues connects the WW and catalytic domain. To characterize the structure and dynamics of full-length Pin1 in solution, high resolution NMR methods have been used to map the nature of interactions between the two domains of Pin1. In addition, the influence of target peptides on domain interactions has been investigated. The studies reveal a dynamic picture of the domain interactions. 15N spin relaxation data, differential chemical shift mapping, and residual dipolar coupling data indicate that Pin1 can either behave as two independent domains connected by the flexible linker or as a single intact domain with some amount of hinge bending motion depending on the sequence of the bound peptide. The functional importance of the modulation of relative domain flexibility in light of the multitude of interaction partners of Pin1 is discussed.     

γ-Aminobutyric acid type A (GABAA) receptor activation modulates tau phosphorylation

Abnormal phosphorylation and aggregation of the microtubule-associated protein Tau are hallmarks of various neurodegenerative diseases, such as Alzheimer disease. Molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. We have developed a novel live cell reporter system based on protein-fragment complementation assay to study dynamic changes in Tau phosphorylation status. In this assay, fusion proteins of Tau and Pin1 (peptidyl-prolyl cis-trans-isomerase 1) carrying complementary fragments of a luciferase protein serve as a sensor of altered protein-protein interaction between Tau and Pin1, a critical regulator of Tau dephosphorylation at several disease-associated proline-directed phosphorylation sites. Using this system, we identified several structurally distinct GABA(A) receptor modulators as novel regulators of Tau phosphorylation in a chemical library screen. GABA(A) receptor activation promoted specific phosphorylation of Tau at the AT8 epitope (Ser-199/Ser-202/Thr-205) in cultures of mature cortical neurons. Increased Tau phosphorylation by GABA(A) receptor activity was associated with reduced Tau binding to protein phosphatase 2A and was dependent on Cdk5 but not GSK3β kinase activity.     

Interactions between protein kinase CK2 and Pin1. Evidence for phosphorylation-dependent interactions

The peptidyl-prolyl isomerase Pin1 interacts in a phosphorylation-dependent manner with several proteins involved in cell cycle events. In this study, we demonstrate that Pin1 interacts with protein kinase CK2, an enzyme that generally exists in tetrameric complexes composed of two catalytic CK2 alpha and/or CK2 alpha' subunits together with two regulatory CK2 beta subunits. Our results indicate that Pin1 can interact with CK2 complexes that contain CK2 alpha. Furthermore, Pin1 can interact directly with the C-terminal domain of CK2 alpha that contains residues that are phosphorylated in vitro by p34(Cdc2) and in mitotic cells. Substitution of the phosphorylation sites of CK2 alpha with alanines resulted in decreased interactions between Pin1 and CK2. The other catalytic isoform of CK2, designated CK2 alpha', is not phosphorylated in mitotic cells and does not interact with Pin1, but a chimeric protein consisting of CK2 alpha' with the C terminus of CK2 alpha was phosphorylated in mitotic cells and interacts with Pin1, further implicating the phosphorylation sites in the interaction. In vitro, Pin1 inhibits the phosphorylation of Thr-1342 on human topoisomerase II alpha by CK2. Topoisomerase II alpha also interacts with Pin1 suggesting that the effect of Pin1 on the phosphorylation of Thr-1342 could result from its interactions with CK2 and/or topoisomerase II alpha. As compared with wild-type Pin1, isomerase-deficient and WW domain-deficient mutants of Pin1 are impaired in their ability to interact with CK2 and to inhibit the CK2-catalyzed phosphorylation of topoisomerase II alpha. Collectively, these results indicate that Pin1 and CK2 alpha interact and suggest a possible role for Pin1 in the regulation of topoisomerase II alpha. Furthermore, these results provide new insights into the functional role of the mitotic phosphorylation of CK2 and provide a new mechanism for selectively regulating the ability of CK2 to phosphorylate one of its mitotic targets.     

Acetylation and phosphorylation processes modulate Tau’s binding to microtubules: A molecular dynamics study

The microtubule-associated protein Tau has its normal function impaired when undergoing post-translational modifications. In this work, molecular modelling techniques were used to infer the effects of acetylation and phosphorylation in Tau's overall conformation, electrostatics, and interactions, but mostly in Tau's ability to bind microtubules. Reported harmful Lys sites were mutated by its acetylated form, generating eight different acetylated Tau (aTau) analogues. Similarly, phosphorylation sites found in normal brains and in Alzheimer’s lesioned brains were considered to design phosphorylated Tau (pTau) analogues. All these designed variants were evaluated in intracellular fluid and near a microtubule (MT) model. Our in silico findings demonstrated that the electrostatic changes, due to the absence of positive Lys’ charges in acetylation cases, or the increasingly negative charge in the phosphorylated forms, hamper the association to the MT tubulins in most cases. Post-translational modifications also pose very distinct conformations to the ones described for native Tau, which hinders the microtubule-binding region (MTBR) and turns difficult the expected binding. Our study elucidates important molecular processes behind Tau abnormal function which can inspire novel therapeutics to address Alzheimer’s disease.     

Pinning down proline-directed phosphorylation signaling

The reversible phosphorylation of proteins on serine or threonine residues preceding proline (Ser/Thr-Pro) is a major cellular signaling mechanism. Although it is proposed that phosphorylation regulates the function of proteins by inducing a conformational change, there are few clues about the actual conformational changes and their importance. Recent identification of the novel prolyl isomerase Pin1 that specifically isomerizes only the phosphorylated Ser/Thr-Pro bonds in certain proteins led us to propose a new signaling mechanism, whereby prolyl isomerization catalytically induces conformational changes in proteins following phosphorylation to regulate protein function. Emerging data indicate that such conformational changes have profound effects on catalytic activity, dephosphorylation, protein-protein interactions, subcellular location and/or turnover. Furthermore, this post-phosphorylation mechanism might play an important role in cell growth control and diseases such as cancer and Alzheimer's.