Cysteine oxidation of tau and microtubule-associated protein-2 by peroxynitrite: modulation of microtubule assembly kinetics by the thioredoxin reductase system

Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative conditions including Alzheimer and Parkinson diseases. We report that peroxynitrite- and hydrogen peroxide-induced disulfides in the neuron-specific microtubule-associated proteins tau and microtubule-associated protein-2 are substrates for the ubiquitous thioredoxin reductase system composed of thioredoxin reductase, human or Escherichia coli thioredoxin, and NADPH. Tau and microtubule-associated protein-2 cysteine oxidation and reduction were quantitated by monitoring the incorporation of 5-iodoacetamidofluorescein, a thiol-specific labeling reagent. Cysteine oxidation of tau and microtubule-associated protein-2 to disulfides altered the ability of the proteins to promote the assembly of microtubules from purified porcine tubulin. Treatment of tau and microtubule-associated protein-2 with either the thioredoxin reductase system or small molecule reductants fully restores the ability of the MAPs to promote microtubule assembly. Thus changes in the redox state of microtubule-associated proteins may regulate microtubule polymerization in vivo.     

Redox modulation of tau and microtubule-associated protein-2 by the glutathione/glutaredoxin reductase system

Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative diseases including Alzheimer's and Parkinson's. We report that peroxynitrite and H2O2-induced disulfides in the porcine brain microtubule-associated proteins tau and microtubule-associated protein-2 are substrates for the glutaredoxin reductase system composed of glutathione reductase, human or Escherichia coli glutaredoxin, reduced glutathione, and NADPH. Oxidation and reduction of cysteines in tau and microtubule-associated protein-2 were quantitated by monitoring the incorporation of 5-iodoacetamido-fluorescein, a thiol-specific labeling reagent. Reduction of disulfide bonds in the microtubule-associated proteins by the glutaredoxin reductase system restored their ability to promote the assembly of microtubules composed of purified porcine tubulin. Thiol-disulfide exchange between oxidized glutathione and the microtubule-associated proteins was detected by monitoring protein oxidation and was quantitated by measuring reduced glutathione by HPLC.     

Interleukin-1 represents a new modality for the activation of extracellular signal-regulated kinases/microtubule-associated protein-2 kinases.

In this study we describe the activation of a protein kinase which phosphorylates a peptide, T669, comprising amino acids 663-681 of the epidermal growth factor receptor and containing the phosphate acceptor site Pro-Leu-Thr669-Pro. In the human epidermoid carcinoma cell line KB, T669 kinase activity in cytosolic extracts peaked (up to 15-fold compared with basal levels) 15-30 min after addition of interleukin-1 (IL-1) and closely paralleled receptor occupancy with a half-maximally effective concentration of approximately 100 pM IL-1 alpha. IL-1 treatment elevated T669 kinase activity to a variable extent in selected fibroblast lines, the hepatoma cell line HepG2, and the murine thymoma EL4 6.1. An IL-1 receptor-negative EL4 variant and the B cell lines 70Z/3, CB23, and RPMI 1788 did not respond in this way. All of the cell lines except 70Z/3 showed increased levels of T669 kinase when treated with the protein kinase C activator phorbol myristate acetate and/or with epidermal growth factor. This finding is in agreement with a previous study (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Activators of protein kinase A did not mimic the ability of IL-1 to stimulate T669 kinase activity, nor did the protein kinase C inhibitor staurosporine abrogate the effect of IL-1. T669 kinase activity from IL-1-stimulated KB cells was partially purified by ion exchange, hydrophobic interaction, and size exclusion chromatography. The partially purified enzyme phosphorylated myelin basic protein, a characteristic substrate of microtubule-associated protein-2 kinase (MAP-2 kinase) and the peptide Arg-Arg-Arg-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4 from RNA polymerase II. Western blotting of chromatographic fractions revealed that T669 kinase activity corresponded with two proteins of 43 and 45 kilodaltons which cross-reacted with antibodies raised against peptide sequences of rat extracellular signal-regulated kinase-1/microtubule-associated protein-2 kinase. T669 kinase activity was critically dependent on the presence of phosphatase inhibitors. Since both the 43- and 45-kDa proteins, immunoprecipitated from [32P]phosphate-labeled cells, demonstrated a dramatic increase in their levels of serine, threonine, and tyrosine phosphorylation after brief treatment with IL-1, we conclude that IL-1 modulates the activity of these extracellular signal-regulated kinase/microtubule-associated protein-2 kinases by altering the level of their phosphorylation.     

An insulin-stimulated ribosomal protein S6 kinase from rabbit liver

In this report we describe an activated form of S6 protein kinase in rabbits treated acutely with insulin. The major insulin-stimulated activity in rabbit liver is increased 2- to 5-fold compared to material from untreated animals based on DEAE-cellulose profiles. The activity observed in DEAE-cellulose fractions can be separated into a major and a minor peak, each having very similar chromatographic behavior. Chromatography on DEAE-cellulose, S-Sepharose, heptyl-Sepharose, heparin-agarose, and Mono Q results in greater than 20,000-fold purification of the insulin-stimulated enzyme with a 12% recovery. The stimulated activity has chromatographic properties similar to an S6 protein kinase studied previously in 3T3-L1 cells (Cobb, M. H. (1986) J. Biol. Chem. 261, 12994-12999) and other systems. The enzyme purified from insulin-treated animals contains a major band that migrates in sodium dodecyl sulfate-polyacrylamide gels with Mr congruent to 70,000; this band also appears in the control preparation. Treatment of the insulin-stimulated S6 kinase with the catalytic subunit of phosphatase 2a reduces its activity by 97%. The activity of the inactivated S6 kinase is stimulated nearly 5-fold by a 15-min preincubation with partially purified insulin-stimulated microtubule-associated protein-2 kinase.     

Proteomic identification of novel substrates of a protein isoaspartyl methyltransferase repair enzyme

We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein l-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1(-/-)) accumulate more isoaspartyl residues than their wild-type littermates, with the most "damaged" residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1(-/-) mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-(3)H]methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and two-dimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1(-/-) animals compared with Pcmt1(+/+) littermates. Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxyl-terminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, beta-synuclein, and alpha-synuclein, are all substrates for the l-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1(-/-) brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice.     

Ascorbic acid reduction of microtubule protein disulfides and its relevance to protein S-nitrosylation assays

The biotin switch assay was developed to aid in the identification of S-nitrosylated proteins in different cell types. However, our work with microtubule proteins including tubulin and its associated proteins tau and microtubule-associated protein-2 shows that ascorbic acid is not a selective reductant of protein S-nitrosothiols as described in the biotin switch assay. Herein we show that ascorbic acid reduces protein disulfides in tubulin, tau, and microtubule-associated protein-2 that are formed by peroxynitrite anion. Reduction of microtubule-associated protein disulfides by ascorbic acid following peroxynitrite treatment restores microtubule polymerization kinetics to control levels. We also show that ascorbic acid reduces the disulfide dithiobis(2-nitrobenzoic acid), a reagent commonly used to detect protein thiols. Not only do we describe a new reactivity of ascorbic acid with microtubule proteins but we expose an important limitation when using the biotin switch assay to detect protein S-nitrosylation.     

p42 MAP kinase phosphorylation sites in microtubule-associated protein tau are dephosphorylated by protein phosphatase 2A1. Implications for Alzheimer's disease [corrected]

The paired helical filament (PHF), which comprises the major fibrous element of the neurofibrillary tangle of Alzheimer's disease, is composed of abnormally phosphorylated microtubule-associated protein tau. Here we show that p42 MAP kinase phosphorylates recombinant tau and converts it to a form which is similar to PHF tau. Of the major serine/threonine protein phosphatases found in mammalian tissues only protein phosphatase 2A (PP2A) could dephosphorylate tau phosphorylated in this manner, with PP2A1 being the most effective form of the enzyme.     

Activation of a Microtubule-Associated Protein-2 Kinase by Insulin-Like Growth Factor-I in Bovine Chromaffin Cells

Treatment of bovine chromaffin cells with insulin-like growth factor-I (IGF-I) caused the activation of a protein kinase that phosphorylates microtubule-associated protein-2 (MAP-2) in vitro. Activation of MAP-2 kinase by IGF-I varied with the time of treatment (maximal at 10-15 min) and the concentration of IGF-I (maximal at 10 nM). The IGF-I-activated MAP-2 kinase was localized to the soluble fraction of chromaffin cell extracts and required Mg2+ for activity. The IGF-I-activated kinase also phosphorylated myelin basic protein, but had little or no activity toward histones or ribosomal S6 protein. To examine the role of protein tyrosine phosphorylation in the activation of the MAP-2 kinase, we isolated phosphotyrosine (PTyr)-containing proteins from chromaffin cells by immunoaffinity adsorption on anti-PTyr-Sepharose beads. Anti-PTyr-Sepharose eluates from IGF-I-treated cells showed increased MAP-2 kinase activity; thus, the MAP-2 kinase (or a closely associated protein) appears to be a PTyr-containing protein. Treatment of anti-PTyr-Sepharose eluates or crude chromaffin cell extracts with alkaline phosphatase significantly decreased kinase activity toward myelin basic protein, indicating that phosphorylation of the IGF-I-activated kinase is required for its activity.     

A novel microtubule-associated protein-2 expressed in oligodendrocytes in multiple sclerosis lesions

Elucidation of the mechanisms involved in the regeneration of oligodendrocytes and remyelination is a central issue in multiple sclerosis (MS) research. We recently identified a novel alternatively spliced, developmentally regulated oligodendrocyte-specific protein designated microtubule-associated protein-2+13 [microtubule-associated protein-2 expressing exon 13 (MAP-2+13)]. MAP-2+13 is expressed in human fetal oligodendrocytes during process extension and myelination but is minimally expressed in normal mature CNS. To test the hypothesis that MAP-2+13 is reexpressed in regenerating oligodendrocytes in MS lesions, we examined the brains of MS patients for the expression of this protein. By immunocytochemistry using a series of monoclonal antibodies specific for MAP-2+13, we determined that MAP-2+13 expression was up-regulated in all 31 lesions from 10 different MS brains. MAP-2+13 was expressed in regenerating oligodendrocytes associated with demyelinated lesions, with the highest counts found in regions of extensive remyelination. By electron microscopy, MAP-2+13 was localized to oligodendrocytes engaged in remyelination, evident by their process extension and association with thinly myelinated (remyelinated) and demyelinated axons. These results suggest a hitherto unsuspected role for this microtubule-associated protein in oligodendrocyte function during development and myelin repair.     

In vivo activation of a microtubule-associated protein kinase during meiotic maturation of the Xenopus oocyte

We have characterized a serine/threonine protein kinase from Xenopus metaphase-II-blocked oocytes, which phosphorylates in vitro the microtubule-associated protein 2 (MAP2). The MAP2 kinase activity, undetectable in prophase oocytes, is activated during the progesterone-induced meiotic maturation (G2-M transition of the cell cycle). p-Nitrophenyl phosphate, a phosphatase inhibitor, is required to prevent spontaneous deactivation of the MAP2 kinase in crude preparations; conversely, the partially purified enzyme can be in vitro deactivated by the low-Mr polycation-stimulated (PCSL) phosphatase (also termed protein phosphatase 2A2), working as a phosphoserine/phosphothreonine-specific phosphatase and not as a phosphotyrosyl phosphatase indicating that phosphorylation of serine/threonine is necessary for its activity. S6 kinase, a protein kinase activated during oocyte maturation which phosphorylates in vitro ribosomal protein S6 and lamin C, can be deactivated in vitro by PCSL phosphatase. S6 kinase from prophase oocytes can also be activated in vitro in fractions known to contain all the factors necessary to convert pre-M-phase-promoting factor (pre-MPF) to MPF. Active MAP2 kinase can activate in vitro the inactive S6 kinase present in prophase oocytes or reactivate S6 kinase previously inactivated in vitro by PCSL phosphatase. These data are consistent with the hypothesis that the MAP2 kinase is a link of the meiosis signalling pathway and is activated by a serine/threonine kinase. This will lead to the regulation of further steps in the cell cycle, such as microtubular reorganisation and S6 kinase activation.     

Characterization of bovine brain calmodulin-dependent protein phosphatase

Calmodulin-dependent protein phosphatase of bovine brain exhibited a pH optimum of 7 and appeared to require sulfhydryl groups for activity. Phosphatase activity was inhibited by both NaF and ZnCl2, but was stimulated approximately 2-fold by MnCl2. The enzyme exhibited broad substrate specificity, dephosphorylating casein, troponin I, protamine, histone, and phosvitin, and was not phosphorylated by cAMP-dependent protein kinase. With 32P-labeled casein as a substrate, phosphatase was activated 15-fold by calmodulin; the dissociation constant of phosphatase for calmodulin was 30 nM. Activation of the enzyme by calmodulin as a function of Ca2+ was highly cooperative; the Hill coefficient was 4.9. At a saturating concentration of calmodulin, half-maximal activation of phosphatase was obtained at 0.3 microM Ca2+. Calmodulin increased the Vmax from 1.7 to 41 nmol mg protein-1 min-1 with no significant change in its Km. Formation of a Ca2+-dependent complex between calmodulin and the phosphatase was demonstrated by a calmodulin-Sepharose affinity column, gel-filtration chromatography, and sedimentation on a sucrose density gradient. The rate of formation and dissociation of the calmodulin X phosphatase complex was rapid and readily reversible in response to changes in Ca2+ concentration. The calmodulin X phosphatase complex consists of 1 mol of calmodulin and 1 mol of phosphatase.     

Characterization of a Nerve Growth Factor-Stimulated Protein Kinase in PC12 Cells Which Phosphorylates Microtubule-Associated Protein 2 and pp250

Treatment of PC12 cells with nerve growth factor (NGF) resulted in the rapid, but transient, activation of a protein kinase which specifically phosphorylated an endogenous 250-kDa cytoskeletal protein (pp250). We report that the microtubule-associated protein, MAP2, is an alternative substrate for the NGF-activated kinase. NGF treatment maximally activated the kinase within 5 min; however, the activity declined with longer exposure to NGF. The enzyme was localized predominantly in microsomal and soluble fractions and phosphorylated MAP2 on serine and threonine residues. The soluble enzyme was fractionated by DEAE chromatography and gel filtration and had an apparent Mr of 45,000. The enzyme was purified to near homogeneity by chromatofocussing and had a pI of 4.9. Kinetic analysis revealed that NGF treatment caused a sevenfold increase in Vmax for MAP2. The Km with respect to the MAP2 substrate was approximately 50 nM and was not altered by NGF treatment. A novel feature of the NGF-stimulated enzyme was its sharp dependence on Mn2+ concentration. The active enzyme is likely to be phosphorylated, because inclusion of phosphatase inhibitors was required for recovery of optimal activity and the activity was lost on treatment of the enzyme with alkaline phosphatase. Histones, tubulin, casein, bovine serum albumin, and the ribosomal subunit protein S-6 were not phosphorylated by this enzyme. The NGF-stimulated kinase was distinct from A kinase, C kinase, or other NGF-stimulated kinases. The rapid and transient activation of the protein kinase upon NGF treatment suggests that the enzyme may play a role in signal transduction in PC12 cells.     

Characterization of Ca2+/Calmodulin-Dependent Protein Kinase Associated with Rat Cerebral Synaptic Junction: Substrate Specificity and Effect of Autophosphorylation

The Ca2+/calmodulin (CaM)-dependent protein kinase associated with rat cerebral synaptic junction (SJ) was characterized, using the SJ fraction as the enzyme preparation, to clarify the functional significance of the enzyme in situ. The protein kinase was greatly activated in the presence of micromolar concentrations of both Ca2+ and calmodulin (EC50 for Ca2+, 1.0 microM; that for CaM, 100 nM). The Km for ATP was 150 microM. SJ proteins were phosphorylated without a lag time, and the phosphorylation reached its maximum within 2-10 min at 25 degrees C. The endogenous substrates consisted of four major (160K, 120K, 60K, and 51K Mr) and 10 minor proteins. Compared with the endogenous substrate phosphorylation, the phosphorylation of exogenously added proteins (myosin light chains from chicken muscle, casein, arginine-rich histone, microtubule-associated protein-2, tau-protein, and tubulin) was weak, although they are expected to be good substrates for the soluble form of the Ca2+/CaM-dependent protein kinase. Autophosphorylation of the enzyme in SJ inhibited its activity and did not alter the subcellular distribution of the enzyme.     

The guanylate kinase domain of the MAGUK PSD-95 binds dynamically to a conserved motif in MAP1a

The postsynaptic density protein PSD-95 and related membrane-associated guanylate kinases are scaffolding proteins, whose modular interaction motifs organize protein complexes at cell junctions. The signature guanylate kinase domain (GK) contains elements of the protein's GMP-binding site but does not bind nucleotide. Instead, the GK domain has evolved from an enzyme to a protein-protein interaction motif. Here, we show that this canonical GMP-binding region interacts with microtubule-associated protein-1a (MAP1a) and we present a structural model. We determine the consensus GK-binding sequence in MAP1a and demonstrate that PSD-95 can use a similar interaction mode to bind diverse protein partners. Furthermore, we show that PSD-95 GK has adopted the conformational flexibility of the ancestral enzyme to bind its varied ligands, which suggests a mechanism of regulation.     

Cyclic modulation of cross-linking interactions of microtubule-associated protein-2 with actin and microtubules by protein kinase FA

The ATP.Mg-dependent type-1 protein phosphatase activating factor (factor FA) was identified as a brain protein kinase that could phosphorylate microtubule-associated protein-2 (MAP-2) and thereby inhibit cross-linking interactions of MAP-2 with actin filaments and microtubules isolated from porcine brain. The phosphorylation sites were found to be equally located on both projection and microtubule-binding domains of MAP-2. Phosphoamino acid analysis revealed that the phosphorylation sites were on both serine and threonine residues, indicating that factor FA is a serine/threonine-specific MAP-2 kinase. Conversely, factor FA was further identified as a MAP-2 phosphatase activator that could promote the dephosphorylation of³²P-MAP-2 phosphorylated by factor FA itself and thereby potentiate cross-linking interactions of MAP-2 with actin and microtubules. Furthermore, the two opposing functions of factor FA can be selectively modulated in a reciprocal manner bypH change. For instance, alkalinepH could stimulate factor FA to work as a MAP-2 kinase but simultaneously block it to work as a MAP-2 phosphatase activator to potentiate the inhibition on the cross-linking interactions of MAP-2 with actin and microtubules. Taken together, the results provide initial evidence that a cyclic modulation of cross-linking interactions of MAP-2 with actin filaments and microtubules can be controlled by factor FA, representing an efficient cyclic cascade control mechanism for rapid structural and functional regulation of neuronal cytoskeletal system.     

The sites at which brain microtubule-associated protein 2 is phosphorylated in vivo differ from those accessible to cAMP-dependent kinase in vitro

The most conspicuous brain microtubule-associated protein, MAP-2, has been shown to contain 8-10 mol of covalently bound phosphate/mol, as isolated. The MAP-2-associated cAMP-dependent protein kinase can add 10-12 more phosphates, using cycled microtubule preparations, but it does not catalyze exchange between ATP and the pre-existing protein phosphate. We now show that the phosphates that turn over in vivo, after intracerebral injection of 32Pi, are primarily in the projection domain of MAP-2, whereas the sites phosphorylated in vitro are more concentrated in the binding domain. Phosphoserine and phosphothreonine were recovered in a 6:1 ratio from partial acid hydrolysates of MAP-2 phosphorylated either in vivo or in vitro. A protein phosphatase, purified from brain, released residues from in vitro sites in both domains. The enzyme did not release appreciable phosphate that had turned over in vivo, and similar specificity was shown by three other purified protein phosphatases: calcineurin (also from brain) and smooth muscle phosphatases I and II. Thus, even in the projection domain, different sites may be involved.     

Identification of a novel protein phosphatase catalytic subunit by cDNA cloning

A cDNA encoding a novel protein phosphatase catalytic subunit (protein phosphatase X) has been isolated from a rabbit liver library. It codes for a protein having 45% and 65% amino acid sequence identity, respectively, to the catalytic subunits of protein phosphatase 1 and protein phosphatase 2A from skeletal muscle. The enzyme is neither the hepatic form of protein phosphatase 1 or 2A, nor is it protein phosphatase 2B or 2C. The possible identity of protein phosphatase X is discussed.     

Gelsolin in Cerebrospinal Fluid as a Potential Biomarker of Epilepsy

Gelsolin is an actin regulatory protein that generally distributed in a wide variety of body tissues, especially the brain tissues and cerebrospinal fluid. In this study we found that lumbar CSF-gelsolin concentrations markedly decreased in epileptic patients by enzyme linked immunosorbent assay. In order to help judge the result, we determined gelsolin expression in temporal lobe tissues of patients with temporal lobe epilepsy using double-label immunofluorescence to location and using western blot to quantitation. Then we observed that gelsolin was co-expressed with microtubule-associated protein-2 in axons and cytoplasms of neurons and gelsolin protein level was also down-regulated in temporal lobe tissues of epileptic patients. Our findings suggested that CSF-gelsolin level might reflect the alteration of gelsolin in brain tissue of epileptic patients and CSF-gelsolin seems to be a potential biomarker for epilepsy.     

Activation of NMDA receptors induces rapid dephosphorylation of the cytoskeletal protein MAP2

Hippocampal slices were preincubated with 32P-orthophosphate and used to study the effect of glutamate analogs on protein phosphorylation. NMDA induced a rapid, 70% decrease in the phosphorylation of the microtubule-associated protein MAP2, with no change in the total amount of MAP2. Both competitive and noncompetitive NMDA antagonists blocked the effect of NMDA, but a glutamate antagonist acting at non-NMDA receptors did not. Kainate and quisqualate were less potent than NMDA in stimulating dephosphorylation of MAP2. Other forebrain regions (necortex, striatum, and olfactory bulb) also showed dephosphorylation of MAP2 in response to NMDA. These and other results suggest that NMDA receptor activation induces the dephosphorylation of MAP2 by stimulating a protein phosphatase, possibly the calcium/calmodulin-dependent protein phosphatase calcineurin. Moreover, they indicate that alteration in the properties of a microtubule-associated protein may account for some of the effects of glutamate on postsynaptic neurons.