Inhibition of PP-2A upregulates CaMKII in rat forebrain and induces hyperphosphorylation of tau at Ser 262/356

The regulation of the activity of CaMKII by PP-1 and PP-2A, as well as the role of this protein kinase in the phosphorylation of tau protein in forebrain were investigated. The treatment of metabolically active rat brain slices with 1.0 microM okadaic acid (OA) inhibited approximately 65% of PP-2A and had no significant effect on PP-1 in the 16000xg tissue extract. Calyculin A (CL-A), 0.1 microM under the same conditions, inhibited approximately 50% of PP-1 and approximately 20% of PP-2A activities. In contrast, a mixture of OA and CL-A practically completely inhibited both PP-2A and PP-1 activities. The inhibition of the two phosphatase activities or PP-2A alone resulted in an approximately 2-fold increase in CaMKII activity and an approximately 8-fold increase in the phosphorylation of tau at Ser 262/356 in 60 min. Treatment of the brain slices with KN-62, an inhibitor of the autophosphorylation of CaMKII at Thr 286/287, produced approximately 60% inhibition in CaMKII activity and no significant effect on tau phosphorylation at Ser 262/356. The KN-62-treated brain slices when further treated with OA and CL-A did not show any change in CaMKII activity. In vitro, both PP-2A and PP-1 dephosphorylated tau at Ser 262/356 that was phosphorylated with purified CaMKII. These studies suggest (i) that in mammalian forebrain the cytosolic CaMKII activity is regulated mainly by PP-2A, (ii) that CaMKII is the major tau Ser 262/356 kinase in brain, and (iii) that a decrease in PP-2A/PP-1 activities in the brain leads to hyperphosphorylation of tau not only by inhibition of its dephosphorylation but also by promoting the CaMKII activity.     

Phosphorylation of tau protein by recombinant GSK-3beta: pronounced phosphorylation at select Ser/Thr-Pro motifs but no phosphorylation at Ser262 in the repeat domain.

Glycogen synthase kinase-3beta (GSK-3beta) has been described as a proline-directed kinase which phosphorylates tau protein at several sites that are elevated in Alzheimer paired helical filaments. However, it has been claimed that GSK-3beta can also phosphorylate the non-proline-directed KXGS motifs in the presence of heparin, including Ser262 in the repeat domain of tau, which could induce the detachment of tau from microtubules. We have analyzed the activity of recombinant GSK-3beta and of GSK-3beta preparations purified from tissue, using two-dimensional phosphopeptide mapping, immunoblotting with phosphorylation-sensitive antibodies, and phosphopeptide sequencing. The most prominent phosphorylation sites on tau are Ser396 and Ser404 (PHF-1 epitope), Ser46 and Thr50 in the first insert, followed by a less efficient phosphorylation of other Alzheimer phosphoepitopes (antibodies AT-8, AT-270, etc). We also show that the non-proline-directed activity at KXGS motifs is not due to GSK-3beta itself, but to kinase contaminations in common GSK-3beta preparations from tissues which are activated upon addition of heparin.     

Luteolin Reduces Zinc-Induced Tau Phosphorylation at Ser262/356 in an ROS-Dependent Manner in SH-SY5Y Cells

In brain, excess zinc alters the metabolism of amyloid precursor protein, leading to ??-amyloid protein deposition, one of the hallmarks of Alzheimer??s disease (AD) pathology. Recently, it has been reported that zinc accelerates in vitro tau fibrillization, another hallmark of AD. In the current study, we examined the effect of high-concentration zinc on tau phosphorylation in human neuroblastoma SH-SY5Y cells. We found that incubation of cells with zinc resulted in abnormal tau phosphorylation at Ser262/356. Moreover, the current study has investigated whether luteolin (Lu), a bioflavonoid, could decrease zinc-induced tau hyperphosphorylation and its underlying mechanisms. Using Western blot and protein phosphatase activity assay, activities of tau kinases and phosphatase were investigated. Our data suggest (1) that zinc induces tau hyperphosphorylation at Ser262/356 epitope and (2) that Lu efficiently attenuates zinc-induced tau hyperphosphorylation through not only its antioxidant action but also its regulation of the phosphorylation/dephosphorylation system.     

Structural characterization of monoclonal antibodies targeting C-terminal Ser404 region of phosphorylated tau protein

Targeting tau with immunotherapies is currently the most common approach taken in clinical trials of patients with Alzheimer’s disease. The most prominent pathological feature of tau is its hyperphosphorylation, which may cause the protein to aggregate into toxic assemblies that collectively lead to neurodegeneration. Of the phospho-epitopes, the region around Ser³⁹⁶/Ser⁴⁰⁴ has received particular attention for therapeutic targeting because of its prominence and stability in diseased tissue. Herein, we present the antigen-binding fragment (Fab)/epitope complex structures of three different monoclonal antibodies (mAbs) that target the pSer⁴⁰⁴ tau epitope region. Most notably, these structures reveal an antigen conformation similar to a previously described pathogenic tau epitope, pSer⁴²², which was shown to have a β-strand structure that may be linked to the seeding core in tau oligomers. In addition, we have previously reported on the similarly ordered conformation observed in a pSer³⁹⁶ epitope, which is in tandem with pSer⁴⁰⁴. Our data are the first Fab structures of mAbs bound to this epitope region of the tau protein and support the existence of proteopathic tau conformations stabilized by specific phosphorylation events that are viable targets for immune modulation.     

alpha-synuclein binds to Tau and stimulates the protein kinase A-catalyzed tau phosphorylation of serine residues 262 and 356.

alpha-Synuclein has been implicated in the pathogenesis of several neurodegenerative disorders based on the direct linking of missense mutations in alpha-synuclein to autosomal dominant Parkinson's disease and its presence in Lewy-like lesions. To gain insight into alpha-synuclein functions, we have investigated whether it binds neuronal proteins and modulates their functional state. The microtubule-associated protein tau was identified as a ligand by alpha-synuclein affinity chromatography of human brain cytosol. Direct binding assays using (125)I-labeled human tau40 demonstrated a reversible binding with a IC(50) about 50 pM. The interacting domains were localized to the C terminus of alpha-synuclein and the microtubule binding region of tau as determined by protein fragmentation and the use of recombinant peptides. High concentrations of tubulin inhibited the binding between tau and alpha-synuclein. Functionally, alpha-synuclein stimulated the protein kinase A-catalyzed phosphorylation of tau serine residues 262 and 356 as determined using a phospho-epitope-specific antibody. We propose that alpha-synuclein modulates the phosphorylation of soluble axonal tau and thereby indirectly affects the stability of axonal microtubules.     

Phosphorylation that detaches tau protein from microtubules (Ser262, Ser214) also protects it against aggregation into Alzheimer paired helical filaments

One of the hallmarks of Alzheimer's disease is the abnormal state of the microtubule-associated protein tau in neurons. It is both highly phosphorylated and aggregated into paired helical filaments, and it is commonly assumed that the hyperphosphorylation of tau causes its detachment from microtubules and promotes its assembly into PHFs. We have studied the relationship between the phosphorylation of tau by several kinases (MARK, PKA, MAPK, GSK3) and its assembly into PHFs. The proline-directed kinases MAPK and GSK3 are known to phosphorylate most Ser-Pro or Thr-Pro motifs in the regions flanking the repeat domain of tau: they induce the reaction with several antibodies diagnostic of Alzheimer PHFs, but this type of phosphorylation has only a weak effect on tau-microtubule interactions and on PHF assembly. By contrast, MARK and PKA phosphorylate several sites within the repeats (notably the KXGS motifs including Ser262, Ser324, and Ser356, plus Ser320); in addition PKA phosphorylates some sites in the flanking domains, notably Ser214. This type of phosphorylation strongly reduces tau's affinity for microtubules, and at the same time inhibits tau's assembly into PHFs. Thus, contrary to expectations, the phosphorylation that detaches tau from microtubules does not prime it for PHF assembly, but rather inhibits it. Likewise, although the phosphorylation sites on Ser-Pro or Thr-Pro motifs are the most prominent ones on Alzheimer PHFs (by antibody labeling), they are only weakly inhibitory to PHF assembly. This implies that the hyperphosphorylation of tau in Alzheimer's disease is not directly responsible for the pathological aggregation into PHFs; on the contrary, phosphorylation protects tau against aggregation.     

A novel brain-specific 25 kDa protein (p25) is phosphorylated by a Ser/Thr-Pro kinase (TPK II) from tau protein kinase fractions

A novel brain-specific 25 kDa protein (p25) was purified from a bovine brain extract. The protein was phosphorylated by Ser/Thr-Pro kinase (TPK II) in tau protein kinase fractions at the Ser residues of Ser-Pro sequences. Using immunoblot analysis, the protein was found only in brain extracts, and was most abundant in the brain regions such as cerebrum and hippocampus, but less abundant in cerebellum, medulla oblongata and olfactory bulb. The protein was detected in rat, bovine and human brain extracts, indicating that this protein specifically exists in mammalian brain tissues.     

Akt/PKB kinase phosphorylates separately Thr212 and Ser214 of tau protein in vitro

Microtubule-associated protein tau contains a consensus motif for protein kinase B/Akt (Akt), which plays an essential role in anti-apoptotic signaling. The motif encompasses the AT100 double phospho-epitope (Thr212/Ser214), a specific marker for Alzheimer's disease (AD) and other neurodegenerations, raising the possibility that it could be generated by Akt. We studied Akt-dependent phosphorylation of tau protein in vitro. We found that Akt phosphorylated both Thr212 and Ser214 in the longest and shortest tau isoforms as determined using phospho site-specific antibodies against tau. Akt did not phosphorylate other tau epitopes, including Tau-1, AT8, AT180, 12E8 and PHF-1. The Akt-phosphorylated tau retained its initial electrophoretic mobility. Immunoprecipitation studies with phospho-specific Thr212 and Ser214 antibodies revealed that only one of the two sites is phosphorylated per single tau molecule, resulting in tau immunonegative for AT100. Mixed kinase studies showed that prior Ser214 phosphorylation by Akt blocked protein kinase A but not GSK3beta activity. On the other hand, GSK3beta selectively blocked Ser214 phosphorylation, which was prevented by lithium. The results suggest that Akt may be involved in AD-specific phosphorylation of tau at the AT100 epitope in conjunction with other kinases. Our data suggest that phosphorylation of tau by Akt may play specific role(s) in Akt-mediated anti-apoptotic signaling, particularly relevant to AD and other neurodegenerations.     

Cerebrospinal fluid tau, A beta, and phosphorylated tau protein for the diagnosis of Alzheimer's disease

The diagnosis of AD is still largely based on exclusion criteria of secondary causes and other forms of dementia with similar clinical pictures, than the diagnostic accuracy of AD is low. Improved methods of early diagnosis are needed, particularly because drugs treatment is more effective in the early stages of the disease. Recent research focused the attention to biochemical diagnostic markers (biomarkers) and according to the proposal of a consensus group on biomarkers, three candidate CSF markers reflecting the pathological AD processes, have recently been identified: total tau protein (t-tau), amyloid beta(1-42) protein (A beta42), and tau protein phosphorylated at AD-specific epitopes (p-tau). Several articles report reduced CSF levels of A beta42 and increased CSF levels of t-tau and p-tau in AD; the sensitivity and specificity of these data are able for discrimination of AD patients from controls. However, the specificity for other dementias is low. According to the literature analysis reported in the present review, we can conclude that the combination of the CSF markers and their ratios may significantly increase the specificity and the accuracy of AD diagnosis.     

Phosphorylation of tau at Ser214 mediates its interaction with 14-3-3 protein: implications for the mechanism of tau aggregation

The microtubule associated protein tau is a major component of neurofibrillary tangles in Alzheimer disease brain, however the neuropathological processes behind the formation of neurofibrillary tangles are still unclear. Previously, 14-3-3 proteins were reported to bind with tau. 14-3-3 Proteins usually bind their targets through specific serine/threonine –phosphorylated motifs. Therefore, the interaction of tau with 14-3-3 mediated by phosphorylation was investigated. In this study, we show that the phosphorylation of tau by either protein kinase A (PKA) or protein kinase B (PKB) enhances the binding of tau with 14-3-3 in vitro . The affinity between tau and 14-3-3 is increased 12- to 14-fold by phosphorylation as determined by real time surface plasmon resonance studies. Mutational analyses revealed that Ser214 is critical for the phosphorylation-mediated interaction of tau with 14-3-3. Finally, in vitro aggregation assays demonstrated that phosphorylation by PKA/PKB inhibits the formation of aggregates/filaments of tau induced by 14-3-3. As the phosphorylation at Ser214 is up-regulated in fetal brain, tau's interaction with 14-3-3 may have a significant role in the organization of the microtubule cytoskeleton in development. Also as the phosphorylation at Ser214 is up-regulated in Alzheimer's disease brain, tau's interaction with 14-3-3 might be involved in the pathology of this disease.     

The development of cell processes induced by tau protein requires phosphorylation of serine 262 and 356 in the repeat domain and is inhibited by phosphorylation in the proline-rich domains

The differentiation of neurons and the outgrowth of neurites depends on microtubule-associated proteins such as tau protein. To study this process, we have used the model of Sf9 cells, which allows efficient transfection with microtubule-associated proteins (via baculovirus vectors) and observation of the resulting neurite-like extensions. We compared the phosphorylation of tau23 (the embryonic form of human tau) with mutants in which critical phosphorylation sites were deleted by mutating Ser or Thr residues into Ala. One can broadly distinguish two types of sites, the KXGS motifs in the repeats (which regulate the affinity of tau to microtubules) and the SP or TP motifs in the domains flanking the repeats (which contain epitopes for antibodies diagnostic of Alzheimer's disease). Here we report that both types of sites can be phosphorylated by endogenous kinases of Sf9 cells, and that the phosphorylation pattern of the transfected tau is very similar to that of neurons, showing that Sf9 cells can be regarded as an approximate model for the neuronal balance between kinases and phosphatases. We show that mutations in the repeat domain and in the flanking domains have opposite effects. Mutations of KXGS motifs in the repeats (Ser262, 324, and 356) strongly inhibit the outgrowth of cell extensions induced by tau, even though this type of phosphorylation accounts for only a minor fraction of the total phosphate. This argues that the temporary detachment of tau from microtubules (by phosphorylation at KXGS motifs) is a necessary condition for establishing cell polarity at a critical point in space or time. Conversely, the phosphorylation at SP or TP motifs represents the majority of phosphate (>80%); mutations in these motifs cause an increase in cell extensions, indicating that this type of phosphorylation retards the differentiation of the cells.     

Rapamycin decreases tau phosphorylation at Ser214 through regulation of cAMP-dependent kinase

Preventing or reducing tau hyperphosphorylation is considered to be a therapeutic strategy in the treatment of Alzheimer’s disease (AD). Rapamycin may be a potential therapeutic agent for AD, because the rapamycin-induced autophagy may enhance the clearance of the hyperphosphorylated tau. However, recent rodent studies show that the protective effect of rapamycin may not be limited in the autophagic clearance of the hyperphosphorylated tau. Because some tau-related kinases are targets of the mammalian target of rapamycin (mTOR), we assume that rapamycin may regulate tau phosphorylation by regulating these kinases. Our results showed that in human neuroblastoma SH-SY5Y cells, treatment with rapamycin induced phosphorylation of the type IIα regulatory (RIIα) subunit of cAMP-dependent kinase (PKA). Rapamycin also induced nuclear translocation of the catalytic subunits (Cat) of PKA and decreases in tau phosphorylation at Ser214 (pS214). The above effects of rapamycin were prevented by pretreatment with the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor U0126. In addition, these effects of rapamycin might not depend on the level of tau expression, because similar results were obtained in both the non-tau-expressing wild type human embryonic kidney 293 (HEK293) cells and HEK293 cells stably transfected with the longest isoform of recombinant human tau (tau441; HEK293/tau441). These findings suggest that rapamycin decreases pS214 via regulation of PKA. Because tau phosphorylation at Ser214 may prime tau for further phosphorylation by other kinases, our findings provide a novel possible mechanism by which rapamycin reduces or prevents tau hyperphosphorylation.     

Laforin, a dual-specificity phosphatase involved in Lafora disease, is phosphorylated at Ser25 by AMP-activated protein kinase

Lafora progressive myoclonus epilepsy [LD (Lafora disease)] is a fatal autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in either the EPM2A gene, encoding the dual-specificity phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Previously, we and others showed that laforin and malin form a functional complex that regulates multiple aspects of glycogen metabolism, and that the interaction between laforin and malin is enhanced by conditions activating AMPK (AMP-activated protein kinase). In the present study, we demonstrate that laforin is a phosphoprotein, as indicated by two-dimensional electrophoresis, and we identify Ser(25) as the residue involved in this modification. We also show that Ser(25) is phosphorylated both in vitro and in vivo by AMPK. Lastly, we demonstrate that this residue plays a critical role for both the phosphatase activity and the ability of laforin to interact with itself and with previously established binding partners. The results of the present study suggest that phosphorylation of laforin-Ser(25) by AMPK provides a mechanism to modulate the interaction between laforin and malin. Regulation of this complex is necessary to maintain normal glycogen metabolism. Importantly, Ser(25) is mutated in some LD patients (S25P), and our results begin to elucidate the mechanism of disease in these patients.     

First structural glimpse at a bacterial Ser/Thr protein phosphatase

In this issue of Structure, we describe the crystal structure of the Ser/Thr protein phosphatase PstP from Mycobacterium tuberculosis, opening new perspectives to understand the putative roles of "eukaryotic-like" signaling elements in bacteria.     

Characterization of the aggregation-prevention activity of p97/valosin-containing protein

The 97 kDa valosin-containing protein (VCP) belongs to a highly conserved AAA (ATPases associated with a variety of activities) family and contains two ATPase domains, D1 and D2. VCP participates in numerous cellular activities, such as membrane fusion, postmitotic Golgi reassembly, endoplasmic reticulum-associated degradation, ubiquitin-proteasome-mediated proteolysis, and many others. In performing these activities, VCP presumably acts as a molecular chaperone that prevents protein aggregation and modifies protein conformation. In this study, we characterized the aggregation-prevention activity of VCP and identified the structural requirement for this activity. We used multiple methods to treat aggregation-prone luciferase (Luc) and showed that VCP prevents the aggregation of Luc in vitro. These results are in agreement; in vivo RNA interference analyses showed that a reduction of VCP level results in more aggregation of Luc in cells. Structural and functional analyses further demonstrated that the D1 domain of VCP is sufficient to mediate the aggregation-prevention activity, which does not require ATP binding, ATP hydrolysis, or a hexameric structure of VCP. Together, these results indicate that (1) VCP prevents protein aggregation in vitro and in vivo, (2) this aggregation-prevention activity is mediated mainly through the D1 domain of VCP, and (3) this activity does not require ATPase activity or a hexameric structure of VCP.     

CGI-58/ABHD5 is phosphorylated on Ser239 by protein kinase A: control of subcellular localization

CGI-58/ABHD5 coactivates adipose triglyceride lipase (ATGL). In adipocytes, CGI-58 binds to perilipin 1A on lipid droplets under basal conditions, preventing interaction with ATGL. Upon activation of protein kinase A (PKA), perilipin 1A is phosphorylated and CGI-58 rapidly disperses into the cytoplasm, enabling lipase coactivation. Because the amino acid sequence of murine CGI-58 has a predicted PKA consensus sequence of RKYS²³⁹S²⁴⁰, we hypothesized that phosphorylation of CGI-58 is involved in this process. We show that Ser239 of murine CGI-58 is a substrate for PKA using phosphoamino acid analysis, MS, and immuno­blotting approaches to study phosphorylation of recombinant CGI-58 and endogenous CGI-58 of adipose tissue. Phosphorylation of CGI-58 neither increased nor impaired coactivation of ATGL in vitro. Moreover, Ser239 was not required for CGI-58 function to increase triacylglycerol turnover in human neutral lipid storage disorder fibroblasts that lack endogenous CGI-58. Both CGI-58 and S239A/S240A-mutated CGI-58 localized to perilipin 1A-coated lipid droplets in cells. When PKA was activated, WT CGI-58 dispersed into the cytoplasm, whereas substantial S239A/S240A-mutated CGI-58 remained on lipid droplets. Perilipin phosphorylation also contributed to CGI-58 dispersion. PKA-mediated phosphorylation of CGI-58 is required for dispersion of CGI-58 from perilipin 1A-coated lipid droplets, thereby increasing CGI-58 availability for ATGL coactivation.     

Phosphorylation of Tau at Thr212, Thr231, and Ser262 Combined Causes Neurodegeneration

Abnormal hyperphosphorylation of the microtubule-associated protein Tau is a hallmark of Alzheimer disease and related diseases called tauopathies. As yet, the exact mechanism by which this pathology causes neurodegeneration is not understood. The present study provides direct evidence that Tau abnormal hyperphosphorylation causes its aggregation, breakdown of the microtubule network, and cell death and identifies phosphorylation sites involved in neurotoxicity. We generated pseudophosphorylated Tau proteins by mutating Ser/Thr to Glu and, as controls, to Ala. These mutations involved one, two, or three pathological phosphorylation sites by site-directed mutagenesis using as backbones the wild type or FTDP-17 mutant R406W Tau. Pseudophosphorylated and corresponding control Tau proteins were expressed transiently in PC12 and CHO cells. We found that a single phosphorylation site alone had little influence on the biological activity of Tau, except Thr²¹², which, upon mutation to Glu in the R406W background, induced Tau aggregation in cells, suggesting phosphorylation at this site along with a modification on the C-terminal of the protein facilitates self-assembly of Tau. The expression of R406W Tau pseudophosphorylated at Thr²¹², Thr²³¹, and Ser²⁶² triggered caspase-3 activation in as much as 85% of the transfected cells, whereas the corresponding value for wild type pseudophosphorylated Tau was 30%. Cells transfected with pseudophosphorylated Tau became TUNEL-positive.     

EGF induces efficient Cx43 gap junction endocytosis in mouse embryonic stem cell colonies via phosphorylation of Ser262, Ser279/282, and Ser368

Gap junctions (GJs) traverse apposing membranes of neighboring cells to mediate intercellular communication by passive diffusion of signaling molecules. We have shown previously that cells endocytose GJs utilizing the clathrin machinery. Endocytosis generates cytoplasmic double-membrane vesicles termed annular gap junctions or connexosomes. However, the signaling pathways and protein modifications that trigger GJ endocytosis are largely unknown. Treating mouse embryonic stem cell colonies – endogenously expressing the GJ protein connexin43 (Cx43) – with epidermal growth factor (EGF) inhibited intercellular communication by 64% and activated both, MAPK and PKC signaling cascades to phosphorylate Cx43 on serines 262, 279/282, and 368. Upon EGF treatment Cx43 phosphorylation transiently increased up to 4-fold and induced efficient (66.4%) GJ endocytosis as evidenced by a 5.9-fold increase in Cx43/clathrin co-precipitation.     

The Ser/Thr/Tyr phosphoproteome of Lactococcus lactis IL1403 reveals multiply phosphorylated proteins

Recent phosphoproteomics studies of several bacterial species have firmly established protein phosphorylation on Ser/Thr/Tyr residues as a PTM in bacteria. In particular, our recent reports on the Ser/Thr/Tyr phosphoproteomes of bacterial model organisms Bacillus subtilis and Escherichia coli detected over 100 phosphorylation events in each of the bacterial species. Here we extend our analyses to Lactococcus lactis, a lactic acid bacterium widely employed by the food industry, in which protein phosphorylation at Ser/Thr/Tyr residues was barely studied at all. Despite the lack of almost any prior evidence of Ser/Thr/Tyr protein phosphorylation in L. lactis, we identified a phosphoproteome of a size comparable to that of E. coli and B. subtilis, with 73 phosphorylation sites distributed over 63 different proteins. The presence of several multiply phosphorylated proteins, as well as over-representation of phosphothreonines seems to be the distinguishing features of the L. lactis phosphoproteome. Evolutionary comparison and the conservation of phosphorylation sites in different bacterial organisms indicate that a majority of the detected phosphorylation sites are species-specific, and therefore have probably co-evolved with the adaptation of the bacterial species to their present-day ecological niches.