Measurement of calpain activity in vitro and in situ using a fluorescent compound and tau as substrates.
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Calpain↗
Biomedical subjects
G V Johnson
Publications and source records attributed to G V Johnson.
The microtubule binding of Tau and high molecular weight Tau in apoptotic PC12 cells is impaired because of altered phosphorylation.
Although the importance of the microtubule network throughout cell life is well established, the dynamics of microtubules during apoptosis, a regulated cell death process, is unclear. In a previous study (Davis, P. K., and Johnson, G. V. (1999) Biochem. J. 340, 51-58) we demonstrated that the phosphorylation of the microtubule-associated protein tau was increased during neuronal PC12 cell apoptosis. The purpose of this study was to determine whether the increased tau phosphorylation that occurred during apoptosis impaired the microtubule binding capacity of tau. This study is the first demonstration that microtubule-binding by tau and high molecular weight tau is significantly impaired as a result of altered phosphorylation during a naturally occurring process, apoptosis. Furthermore, co-immunofluorescence studies reveal for the first time that tau populations within an apoptotic neuronal PC12 cell exhibit differential phosphorylation. In control PC12 cells, Tau-1 staining (Tau-1 recognizes an unphosphorylated epitope) is evident throughout the entire cell body. In contrast, Tau-1 immunoreactivity in apoptotic PC12 cells is retained in the nuclear/perinuclear region but is significantly decreased in the cytoplasm up to the plasma membrane. The selective distribution of phosphorylated tau in apoptotic PC12 cells indicates that tau likely plays a significant role in the cytoskeletal changes that occur during apoptosis.
Animals↗
Novel bimodal effects of the G-protein tissue transglutaminase on adrenoreceptor signalling.
Tissue transglutaminase (tTG) is a novel G-protein that previous studies showed can couple ligand-bound activated alpha(1B) adrenoreceptors to phospholipase C-delta, resulting in phosphoinositide (PI) hydrolysis. In human neuroblastoma SH-SY5Y cells we found that although endogenous tTG can facilitate alpha(1B) adrenoreceptor-stimulated PI hydrolysis, its contribution is minor compared with the classical heterotrimeric G-protein G(q/11). Further, we show that the alpha(1B) adrenoreceptor recruits tTG to the membrane and that this recruitment is enhanced by agonist occupancy of the receptor. In addition, the effects of tTG on signalling are bimodal. At low expression levels, tTG enhanced alpha(1B) adrenoreceptor-stimulated PI hydrolysis, whereas at higher expression levels tTG attenuated significantly this response. These findings are the first to demonstrate that a protein can both facilitate and attenuate receptor-stimulated PI hydrolysis.
Adrenergic alpha-Agonists↗
Rapid, single-step procedure for the identification of transglutaminase-mediated isopeptide crosslinks in amino acid digests.
Tissue transglutaminase (tTG) is a calcium-activated enzyme which can covalently crosslink the epsilon-amino group of a peptide-bound lysine into the gamma-carboxamide group of a peptide-bound glutamine, forming a epsilon-(-gamma-glutamyl)lysine isopeptide bond. We have developed a sensitive, single-step method for the isolation and detection of tTG-mediated isopeptide bonds from purified proteins and tissue homogenates. This method offers significantly improved resolution over current techniques, and obviates the need for multi-column systems or costly fluorescence monitors. By using enzymatic proteolysis, derivatization with phenylisothiocyanate, and a simple elution gradient for HPLC, we were able to determine the frequency of crosslinks in purified fibrin (1.7 mol of isodipeptide per mol of fibrin), crosslinked tau proteins (0.75 mol of isodipeptide per mol of tau), and whole-tissue liver homogenates (0.5 nmol of isodipeptide per mg of total protein).
Amino Acids↗
Transient increases in intracellular calcium result in prolonged site-selective increases in Tau phosphorylation through a glycogen synthase kinase 3beta-dependent pathway.
Calcium is a universal intracellular signaling molecule. Through variations in both the amplitude and frequency of intracellular calcium increases, the same calcium ion can elicit different responses. In this report, we investigated the effect of a calcium transient, lasting 2-5 min, on alterations in the phosphorylation state of the cytoskeletal protein, tau. Transient increases in calcium result in a prolonged (1-4 h) approximately 60% increase in tau phosphorylation at the Tau-1 epitope. These increases in tau phosphorylation appear to be more dependent upon the duration of the increase in intracellular calcium and less on the amplitude. The calcium-induced increases in tau phosphorylation are not dependent upon protein synthesis, nor are protein kinase C or calcium/calmodulin-dependent protein kinase II involved in the response. However, the calcium-induced increase in tau phosphorylation was inhibited by lithium, a noncompetitive inhibitor of glycogen synthase kinase-3beta (GSK-3beta), and by the tyrosine kinase inhibitor, genistein. Furthermore, transient increases in calcium resulted in a prolonged increase in GSK-3beta tyrosine phosphorylation concomitant with the increase in tau phosphorylation. Therefore, this study is the first to indicate that transient increases in intracellular calcium result in increased tyrosine phosphorylation and activation of GSK-3beta which subsequently results in a sustained increase in the phosphorylation state of tau.
Calcium↗
Energy metabolism and protein phosphorylation during apoptosis: a phosphorylation study of tau and high-molecular-weight tau in differentiated PC12 cells.
Apoptosis has been characterized as a regulated, energy-dependent process. Specific protein-phosphorylation events have been demonstrated previously to occur during apoptosis and may play an important role in the regulation of this death process. In this study, energy metabolism and protein phosphorylation during apoptosis of neuronal PC12 cells induced by nerve growth factor and serum deprivation was examined using [32P]Pi-labelling techniques. Although ATP levels were maintained at control levels during apoptosis, [32P]Pi incorporation into ATP was decreased significantly, coinciding with an almost identical decrease in Na+-dependent phosphate uptake. During neuronal PC12-cell apoptosis, increased phosphorylation of tau and high-molecular-weight (HMW) tau was observed within the epitope of Tau-1, a phosphate-dependent tau antibody that only recognizes the unphosphorylated form of its epitope. In addition, based on two-dimensional phosphopeptide mapping, [32P]Pi incorporation into a phosphopeptide of tau and HMW tau from apoptotic cells increased. Whereas [32P]Pi incorporation into total protein decreased to 23% of the control during apoptosis, [32P]Pi incorporation into tau and HMW tau was significantly higher, indicating a preferential phosphorylation of specific proteins during the apoptotic process. This study provides novel information about phosphate uptake, incorporation of [32P]Pi into ATP, and protein phosphorylation events during apoptosis.
Adenosine Triphosphate↗
Hippocampal microtubule-associated protein-2 alterations with contextual memory.
Using immunohistochemistry and immunoblots, we show that alterations in hippocampal microtubule-associated protein-2 appear to be highly correlated with contextual memory as measured by significantly heightened fear responses. Compared to naive controls, rats trained in a novel context showed significantly increased immunostaining for the high molecular weight microtubule-associated protein-2a/b. This increase was observed 2 weeks after training and it was selective for hippocampal CA1 and CA2 pyramidal cells. Pre-exposure to the training context one month before training altered the hippocampal microtubule-associated protein-2 response; in these animals only the dentate gyrus showed significantly increased microtubule-associated protein-2a/b. Training-related increases in immunohistochemical staining for microtubule-associated protein-2 suggested that there was an increase in overall intact protein, an increase in immunoreactive breakdown products, or changes in protein compartmentalization. Immunoblots of hippocampal homogenates reacted with monoclonal antibodies to microtubule-associated protein-2a/b showed an increased presence of breakdown products in trained animals compared to untrained controls. Additional immunoblot studies demonstrated statistically significant decreases in the levels and/or phosphorylation state of the low molecular weight microtubule-associated protein-2c in the hippocampus of trained animals as compared to that of controls. These alterations in microtubule-associated protein-2 may reflect dendritic remodeling related to contextual memory storage.
Animals↗
Glycogen synthase kinase-3beta, beta-catenin, and tau in postmortem bipolar brain.
Therapeutic concentrations of the anti-bipolar drug lithium inhibit the activity of glycogen synthase kinase-3beta, which raises the possibility that this enzyme and its substrates may be altered in the brain of subjects with bipolar disorder. Therefore, in prefrontal cortical samples from subjects with bipolar disorder and age-matched control subjects, we examined the levels of glycogen synthase kinase 3beta and of two proteins modified by it, beta-catenin and the microtubule associated protein tau. There were no significant differences between subject groups among these measurements, but there was a tendency for the tau isoform profile to be modified in bipolar tissue. Thus, while there are no differences between bipolars and controls in prefrontal cortical levels of glycogen synthase kinase-3beta, beta-catenin, or tau, tau isoform levels or phosphorylation states may be modified in bipolar disorder.
Adult↗
Insulin transiently increases tau phosphorylation: involvement of glycogen synthase kinase-3beta and Fyn tyrosine kinase.
The modulation of tau phosphorylation in response to insulin was examined in human neuroblastoma SH-SY5Y cells. Insulin treatment resulted in a transient increase in tau phosphorylation followed by a decrease in tau phosphorylation that correlated directly with a sequential activation and deactivation of glycogen synthase kinase-3beta (GSK-3beta). The insulin-induced increase in tau phosphorylation and concurrent activation of GSK-3beta was rapid (<2 min) and transient, and was associated with increased tyrosine phosphorylation of GSK-3beta. The increase in GSK-3beta tyrosine phosphorylation corresponded directly to an increase in the association of Fyn tyrosine kinase with GSK-3beta, and Fyn immunoprecipitated from cells treated with insulin for 1 min phosphorylated GSK-3beta to a significantly greater extent than Fyn immunoprecipitated from control cells. Subsequent to the increase in GSK-3beta activation and tau phosphorylation, treatment of cells with insulin for 60 min resulted in a dephosphorylation of tau and a decrease in GSK-3beta activity. Thus, insulin rapidly and transiently activated GSK-3beta and modulated tau phosphorylation, alterations that may contribute to neuronal plasticity.
Calcium-Calmodulin-Dependent Protein Kinases↗
p38 kinase is activated in the Alzheimer's disease brain.
The p38 mitogen-activated protein kinase is a stress-activated enzyme responsible for transducing inflammatory signals and initiating apoptosis. In the Alzheimer's disease (AD) brain, increased levels of phosphorylated (active) p38 were detected relative to age-matched normal brain. Intense phospho-p38 immunoreactivity was associated with neuritic plaques, neuropil threads, and neurofibrillary tangle-bearing neurons. The antibody against phosphorylated p38 recognized many of the same structures as an antibody against aberrantly phosphorylated, paired helical filament (PHF) tau, although PHF-positive tau did not cross-react with the phospho-p38 antibody. These findings suggest a neuroinflammatory mechanism in the AD brain, in which aberrant protein phosphorylation affects signal transduction elements, including the p38 kinase cascade, as well as cytoskeletal components.
Aged↗
Altered protein kinase C activation of Na+/Ca2+ exchange in mesangial cells from salt-sensitive rats.
The purpose of these studies was to determine whether there is a defect in protein kinase C (PKC) regulation of the Na+/Ca2+ exchanger in cultured mesangial cells (MC) from Dahl/Rapp salt-sensitive (S) and salt-resistant (R) rats. R and S MCs were cultured, grown on coverslips, and loaded with fura 2 for measurement of single cell cytosolic calcium concentration ([Ca2+]i) in a microscope-based photometry system. Studies were performed in cells that were exposed to serum (serum fed) and in cells that were serum deprived for 24 h. Baseline [Ca2+]i values measured in a Ringer solution containing 150 mM NaCl were similar between R and S MCs in both serum-fed and serum-deprived groups, although baseline [Ca2+]i values were uniformly higher in the serum-deprived groups. Exchanger activity was assessed by reducing extracellular Na (Nae) from 150 to 2 mM, which resulted in movement of Na+ out of and Ca2+ into these cells (reverse-mode Na+/Ca2+ exchange). PKC was activated in these cells with 15-min exposure to 100 nM phorbol 12-myristate 13-acetate (PMA). In the absence of PMA, the change in [Ca2+]i (Delta[Ca2+]i) with reduction in Nae was similar between R and S MCs in both serum-fed and serum-deprived groups, although the magnitude of Delta[Ca2+]i was enhanced by serum deprivation. In both serum-fed and serum-deprived groups, PMA significantly increased Delta[Ca2+]i in R but not S MCs. Upregulation of exchanger activity in R MCs could be abolished by prior 24-h exposure to PMA, a maneuver that downregulates PKC activity. Other studies were performed to evaluate exchanger protein expression using monoclonal and polyclonal antibodies. Immunoblots of PMA-treated cells revealed an increase in the levels of 70- and 120-kDa proteins in the crude membrane fraction of R but not S MCs, an increase which was abrogated by prior 24-h PMA pretreatment and corresponded to reduction in the 70-kDa protein in the crude cytosolic fraction. These data demonstrate that PKC enhances Na+/Ca2+ exchange activity in MCs from R but not from S rats, suggesting that there may be a defect in the PKC-Na+/Ca2+ exchange regulation pathway in MCs of S rats.
Animals↗
Endoscopically-assisted excision of scapular osteochondroma.
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Adolescent↗
Tau protein in normal and Alzheimer's disease brain.
In 1975, Weingarten and colleagues isolated a protein factor that was able to induce microtubule formation. They called this factor tau (t). Some ten years later a new era of research on this microtubule-associated protein was launched when several groups almost simultaneously discovered that tau was the predominant protein component of the paired helical filaments (PHFs) and neurofibrillary tangles (NFTs) which are characteristic pathological lesions of the Alzheimer's disease brain. Subsequent findings that PHF-tau isolated from Alzheimer's disease brain was phosphorylated to a greater extent than non-PHF tau, led to extensive investigation into the posttranslational modifications (mainly phosphorylation) of tau in normal and Alzheimer's disease brain. The present review highlights the literature concerning the normal functioning and processing of tau protein, and examines the evidence for the involvement of the abnormal posttranslational processing of tau in the pathology of Alzheimer's disease. Finally, speculation as to the relationship between abnormal processing of tau, other subcellular abnormalities seen in Alzheimer's disease, and the pathological causes of the disease are discussed.
Journal Article↗
Tau protein in normal and Alzheimer's disease brain: an update.
Tau is a microtubule-associated protein that, in a hyperphosphorylated form, comprises the main component of the paired helical filaments and neurofibrillary tangles found in Alzheimer's Disease (AD) brain. It is therefore important to understand the normal functioning and processing of tau protein, and the abnormal posttranslational processing of tau in AD pathology. In 1996, Johnson and Jenkins reviewed the literature on the biochemistry, function, and phosphorylation of tau in normal and AD brain. Since that time, numerous publications have come out further elucidating the properties of tau. The present review updates the topics originally covered in the 1996 review, as well as presents a number of new topics. For example, mutations in the tau gene have been found in several non-AD, autosomal dominant neurodegenerative disorders that exhibit extensive neurofibrillary pathology. In addition, there is increasing evidence that tau may be involved in signal transduction, organelle transport, and cell growth, independent of its microtubule-binding functions. Taken together, the research reviewed here demonstrates that tau is a very complex protein with various functions that are intricately regulated. It is clear that more research is required to completely understand the functions and regulation of tau in normal and AD brain.
Journal Article↗
Modulation of tau phosphorylation within its microtubule-binding domain by cellular thiols.
Tau is a microtubule-stabilizing protein that is functionally modulated by alterations in its phosphorylation state. Because phosphorylation regulates both normal and pathological tau functioning, it is of importance to identify the signaling pathways that regulate tau phosphorylation in vivo. The present study examined changes in tau phosphorylation and function in response to modulation of cellular thiol content. Treatment of cells with phenylarsine oxide, which reacts with vicinal thiols, selectively increased tau phosphorylation within its microtubule-binding domain, at the non-Ser/Thr-Pro sites Ser262/356, while decreasing tau phosphorylation at Ser/ Thr-Pro sites outside this region. This increase in tau phosphorylation correlated with a decrease in the amount of tau associated with the cytoskeleton and decreased microtubule stability. Phenylarsine oxide-induced tau phosphorylation was inhibited by oxidants and by the protein kinase inhibitor staurosporine. Although staurosporine completely eliminated the increase in tau phosphorylation at Ser262/356, as detected by immunostaining with 12E8, it had a comparatively minor effect on the changes in tau localization induced by phenylarsine oxide. The results suggest that regulation of cellular thiols is important for modulating tau phosphorylation and function in situ. Additionally, although phosphorylation of Ser262/356 decreases tau's interaction with the cytoskeleton, phosphorylation of these residues alone is not sufficient for the phenylarsine oxide-induced changes in tau localization.
Animals↗
Tau is modified by tissue transglutaminase in situ: possible functional and metabolic effects of polyamination.
Tissue transglutaminase (tTG) is up-regulated in Alzheimer's disease brain and localizes to neurofibrillary tangles with the tau protein. Tau is an in vitro tTG substrate, being cross-linked and/or polyaminated. Further, the Gln and Lys residues in tau that are modified by tTG in vitro are located primarily within or adjacent to the microtubule-binding domains. Considering these and other previous findings, this study was carried out to determine if tau is modified in situ by tTG in human neuroblastoma SH-SY5Y cells, and whether tTG-catalyzed tau polyamination modulates the function and/or metabolism of tau in vitro. For these studies, SH-SY5Y cells stably overexpressing tTG were used. tTG coimmunoprecipitated with tau, and elevating intracellular calcium levels with maitotoxin resulted in a 52 +/- 4% increase in the amount of tTG that coimmunoprecipitated with tau. The increase in association of tTG with tau after treatment with maitotoxin corresponded to a coimmunolocalization of tTG, tTG activity, and tau in the cells. Further, tau was modified by tTG in situ in response to maitotoxin treatment. In vitro polyaminated tau was significantly less susceptible to micro-calpain proteolysis; however, tTG-mediated polyamination of tau did not significantly alter the microtubule-binding capacity of tau. Thus, tau interacts with and is modified by tTG in situ, and modification of tau by tTG alters its metabolism. These data indicate that tau is likely to be modified physiologically and pathophysiologically by tTG, and tTG may play a role in Alzheimer's disease.
Alzheimer Disease↗
Tissue transglutaminase is increased in Huntington's disease brain.
The polyglutamine-expanded N-terminal region of mutant huntingtin causes neurodegeneration in Huntington's disease (HD). Neuronal intranuclear and cytosolic inclusions composed of mutant huntingtin are found in brains of HD patients. Because tissue transglutaminase cross-links proteins into filamentous aggregates and polypeptide-bound glutamines are primary determining factors for tissue transglutaminase-catalyzed reactions, it has been hypothesized that tissue transglutaminase may contribute to the formation of these aggregates. In this report immunohistochemical and biochemical methods were used to demonstrate that tissue transglutaminase expression and transglutaminase activity are elevated in HD brains in a grade-dependent manner. In the striatum, tissue transglutaminase activity was significantly increased in the grade 3 HD cases compared with controls. When normalized to the neuronal marker calbindin D28k, immunoblot analysis revealed that in the striatum the levels of tissue transglutaminase were significantly increased in all HD cases compared with controls. Immunohistochemical staining of the HD striatum revealed that tissue transglutaminase immunoreactivity was markedly increased in all grades as compared with controls. In the superior frontal cortex, tissue transglutaminase activity was significantly higher in all HD cases as compared with controls. Quantitative analysis of immunoblots demonstrated that tissue transglutaminase levels were elevated in HD grades 2 and 3 cases. Tissue transglutaminase immunoreactivity within the superior frontal neocortex was also greater in all the HD cases compared with controls. These data clearly indicate that tissue transglutaminase is elevated in HD brain and may play a role in the disease process.
Aged↗
Enhancement of peroxynitrite-induced apoptosis in PC12 cells by fibroblast growth factor-1 and nerve growth factor requires p21Ras activation and is suppressed by Bcl-2.
Extracellular trophic factors can regulate whether cells subjected to oxidative stress will survive to proliferate or else undergo cell death. We have previously shown that about 35% of undifferentiated PC12 cells undergo apoptosis 18 h after exposure to peroxynitrite and that pretreatment with nerve growth factor (NGF) protects PC12 cells through activation of phosphatidylinositol (PI) 3-kinase. In contrast, pretreatment with acidic fibroblast growth factor (FGF-1) approximately doubled apoptosis. We report here that NGF added immediately after peroxynitrite treatment no longer protected against apoptosis, but instead enhanced apoptosis to the same extent as FGF. We further investigated which signaling pathways were involved in increasing the level of apoptosis. Overexpression of Bcl-2 blocked the increased apoptosis caused by NGF and FGF-1, but Bcl-2 did not prevent the induction of apoptosis by peroxynitrite alone. The increase in apoptosis caused by the trophic factors was also blocked by the expression of a dominant negative p21Ras mutant. Activation of PI 3-kinase by NGF pretreatment completely protected against both the enhanced apoptosis induced by FGF-1 pretreatment and NGF posttreatment and the apoptosis induced by peroxynitrite alone. Our results indicate that the enhancement of peroxynitrite-induced apoptosis caused by NGF and FGF-1 is dependent on the stimulation of a proapoptotic pathway involving p21Ras that can be suppressed by Bcl-2.
Animals↗