Biomedical subjects

G V Johnson

Publications and source records attributed to G V Johnson.

At least 55 records · Page 3Linked to original sources

PubMed1998

Calcineurin inhibition prevents calpain-mediated proteolysis of tau in differentiated PC12 cells.

The effects of calcium influx on tau levels and phosphorylation were examined in differentiated PC12 cells. Maitotoxin-induced calcium influx resulted in time- and concentration-dependent tau dephosphorylation and degradation. Incubation of PC12 cells with a membrane-permeable calpain inhibitor blocked maitotoxin-induced tau degradation, suggesting the involvement of calpain in calcium-stimulated tau turnover. Okadaic acid or the calcineurin inhibitor FK520 partially inhibited maitotoxin-induced tau dephosphorylation at the Tau-1 epitope, indicating both phosphatase 2A/1 and calcineurin were involved. In addition, FK520, but not okadaic acid, blocked the maitotoxin-induced tau degradation, demonstrating that dephosphorylation of specific tau epitopes by was essential for calpain-mediated tau degradation. Moreover, maitotoxin effects were likely independent of tau association with microtubules because maitotoxin induced tau degradation and dephosphorylation in the presence of either nocodazole or taxol. These data provide evidence that calpain is involved in tau turnover in situ and calcineurin plays an important role in modulating tau susceptibility to calpain.

Animals↗

PubMed1998

The interrelationship between selective tau phosphorylation and microtubule association.

The purpose of this study was to examine the modulation of tau phosphorylation mediated by protein kinase A, a kinase with low intrinsic activity, and by the constitutively active glycogen synthase kinase, as well as to examine the subsequent effects on tau-microtubule association in differentiated human SH-SY5Y neuroblastoma cells. Activation of protein kinase A with forskolin and rolipram significantly increased tau phosphorylation at Ser262/356 only in the presence of okadaic acid, indicating that phosphates at these sites are normally turned over rapidly. In contrast, glycogen synthase kinase appears to maintain tau phosphorylation at Thr181 and Ser396/404 since inhibition of glycogen synthase kinase with lithium reduced phosphorylation at these sites. Lithium treatment also significantly decreased tau and tyrosinated alpha-tubulin levels. Perturbation of microtubules with nocodazole or taxol induced tau dephosphorylation at Tau-1 sites, Thr181 and Ser396/404, indicating that both constitutive kinase activity and microtubule state modulate tau phosphorylation at these sites. Nocodazole- or taxol-induced tau dephosphorylation was blocked by the protein phosphatase 2A/1 inhibitor okadaic acid, but not by the protein phosphatase 2B inhibitor cyclosporin A. In addition, osmotic stress, such as treatment with 20 mM NaCl, selectively increased tau phosphorylation at the Tau-1 epitope. To investigate the effect of phosphorylation on tau association with microtubules and microtubule stability in situ, a Triton X-100 extraction assay was utilized to separate the detergent-soluble cytosolic components from the detergent-insoluble cytoskeletal components. In control cells or cells treated with lithium very little tau was detected in the cytosolic fraction. Activation of protein kinase A in the presence of okadaic acid elevated tau levels in the detergent-soluble fraction, which contained all the tau phosphorylated at Ser262/356, and also decreased microtubule stability, as indicated by decreased acetylated alpha-tubulin levels. In conclusion, the phosphorylation state of tau in differentiated SH-SY5Y cells is regulated by glycogen synthase kinase, microtubule dynamics and osmotic stress at overlapping sites which apparently have little influence on tau-microtubule association. In contrast, phosphorylation of tau at Ser262/356 within the microtubule-binding, which was mediated in part by protein kinase A, prevented the association of tau with microtubules in situ.

Amino Acid Sequence↗

PubMed1998

Oxidative stress inhibits calpain activity in situ.

In this study, the effects of oxidative stress on calpain-mediated proteolysis and calpain I autolysis in situ were examined. Calpain activity was stimulated in SH-SY5Y human neuroblastoma cells with the calcium ionophore, ionomycin. Calpain-mediated proteolysis of the membrane-permeable fluorescent substrate N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4-methylcouma rin, as well as the endogenous protein substrates microtubule-associated protein 2, tau and spectrin, was measured. Oxidative stress, induced by addition of either doxorubicin or 2-mercaptopyridine N-oxide, resulted in a significant decrease in the extent of ionophore-stimulated calpain activity of both the fluorescent compound and the endogenous substrates compared with control, normoxic conditions. Addition of glutathione ethyl ester, as well as other antioxidants, resulted in the retention/recovery of calpain activity, indicating that oxidation-induced calpain inactivation was preventable/reversible. The rate of autolytic conversion of the large subunit of calpain I from 80 to 78 to 76 kDa was decreased during oxidative stress; however, the extent of calpain autolysis was not altered. These data indicate that oxidative stress may reversibly inactivate calpain I in vivo.

Calpain↗

PubMed1998

Distinct nuclear localization and activity of tissue transglutaminase.

Tissue transglutaminase is a calcium-dependent transamidating enzyme that has been postulated to play a role in the pathology of expanded CAG repeat disorders with polyglutamine expansions expressed within the affected proteins. Because intranuclear inclusions have recently been shown to be a common feature of many of these codon reiteration diseases, the nuclear localization and activity of tissue transglutaminase was examined. Subcellular fractionation of human neuroblastoma SH-SY5Y cells demonstrated that 93% of tissue transglutaminase is localized to the cytosol. Of the 7% found in the nucleus, 6% copurified with the chromatin-associated proteins, and the remaining 1% was in the nuclear matrix fraction. In situ transglutaminase activity was measured in the cytosolic and nuclear compartments of control cells, as well as cells treated with the calcium-mobilizing agent maitotoxin to increase endogenous tissue transglutaminase activity. These studies revealed that tissue transglutaminase was activated in the nucleus, a finding that was further supported by cytochemical analysis. Immunofluorescence studies revealed that nuclear proteins modified by transglutaminase exhibited a discrete punctate, as well as a diffuse staining pattern. Furthermore, different proteins were modified by transglutaminase in the nucleus compared with the cytosol. The results of these experiments clearly demonstrate localization of tissue transglutaminase in the nucleus that can be activated. These findings may have important implications in the formation of the insoluble nuclear inclusions, which are characteristic of codon reiteration diseases such as Huntington's disease and the spinocerebellar ataxias.

Cell Nucleus↗

PubMed1998

Modulation of the in situ activity of tissue transglutaminase by calcium and GTP.

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the posttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Previous in vitro studies have demonstrated that the transamidating activity of tTG requires calcium and is inhibited by GTP. To investigate the endogenous regulation of tTG, a quantitative in situ transglutaminase (TG) activity assay was developed. Treatment of human neuroblastoma SH-SY5Y cells with retinoic acid (RA) resulted in a significant increase in tTG levels and in vitro TG activity. In contrast, basal in situ TG activity did not increase concurrently with RA-induced increased tTG levels. However, stimulation of cells with the calcium-mobilizing drug maitotoxin (MTX) resulted in increases in in situ TG activity that correlated (r2 = 0.76) with increased tTG levels. To examine the effects of GTP on in situ TG activity, tiazofurin, a drug that selectively decreases GTP levels, was used. Depletion of GTP resulted in a significant increase in in situ TG activity; however, treatment of SH-SY5Y cells with a combination of MTX and tiazofurin resulted in significantly less in situ TG activity compared with treatment with MTX alone. This raised the possibility of calcium-dependent proteolysis due to the effects of tiazofurin, because in vitro GTP protects tTG against proteolysis by trypsin. Studies with a selective membrane permeable calpain inhibitor indicated that tTG is likely to be an endogenous substrate of calpain, and that depletion of GTP increases tTG degradation after elevation of intracellular calcium levels. TG activity was also increased in response to activation of muscarinic cholinergic receptors, which increases intracellular calcium through inositol 1,4,5-trisphosphate generation. The results of these experiments demonstrate that selective changes in calcium and GTP regulate the activity and levels of tTG in situ.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1998

Tau complexes with phospholipase C-gamma in situ.

Based on the results of recent in vitro studies, tau has been proposed to be involved in regulating signal transduction through the phospholipase C-gamma (PLC-gamma) signaling pathway. The present study provides support for the physiological relevance of this hypothesis by demonstrating the existence of a tau-PLC-gamma complex in situ in a human neuroblastoma cell line. Both PLC-gamma and PLC-delta, but not PLC-beta, co-purified with microtubule-associated proteins. PLC-gamma, but neither PLC-delta nor PLC-beta, co-immunoprecipitated with tau, and the PLC co-precipitating with tau was enzymatically active. Additionally, both tau and MAP-2 co-precipitated with PLC-gamma. These studies indicate that tau associates, either directly or indirectly, with PLC-gamma in situ, suggesting that tau may be appropriately localized to participate in the regulation of signal transduction through the PLC-gamma pathway in vivo.

Humans↗

PubMed1998

Tissue transglutaminase is an in situ substrate of calpain: regulation of activity.

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the transamidation of specific polypeptide-bound glutamine residues, a reaction that is inhibited by GTP. There is also preliminary evidence that, in situ, calpain and GTP may regulate tTG indirectly by modulating its turnover by the calcium-activated protease calpain. In the present study, the in vitro and in situ proteolysis of tTG by calpain, and modulation of this process by GTP, was examined. tTG is an excellent substrate for calpain and is rapidly degraded. Previously it has been demonstrated that GTP binding protects tTG from degradation by trypsin. In a similar manner, guanosine-5'-O-(3-thiotriphosphate) protects tTG against proteolysis by calpain. Treatment of SH-SY5Y cells with 1 nM maitotoxin, which increases intracellular calcium levels, resulted in a significant increase in in situ TG activity, with only a slight decrease in tTG protein levels. In contrast, when GTP levels were depleted by pretreating the cells with tiazofurin, maitotoxin treatment resulted in an approximately 50% decrease in tTG protein levels, and a significant decrease in TG activity, compared with maitotoxin treatment alone. Addition of calpain inhibitors inhibited the degradation of tTG in response to the combined treatment of maitotoxin and tiazofurin and resulted in a significant increase in in situ TG activity. These studies indicate that tTG is an endogenous substrate of calpain and that GTP selectively inhibits the degradation of tTG by calpain.

Antineoplastic Agents↗

PubMed1997

Phosphorylation of microtubule-associated protein tau on Ser 262 by an embryonic 100 kDa protein kinase.

This study examined the phosphorylation of tau on Ser 262, within the first microtubule-binding domain, by a developmentally regulated 100 kDa protein kinase exhibiting significantly greater activity in the embryonic rat brain than in the adult rat brain. This protein kinase co-purified with microtubules and co-immunoprecipitated with both tau and MAP-2. In addition to phosphorylating tau, MAP-2, and a Ser 262-containing peptide, the present protein kinase activity was shown to autophosphorylate as determined by the in-gel kinase assay in the absence of any protein or peptide polymerized into the matrix. Phosphorylation of tau with this protein kinase significantly reduced the tau-microtubule interaction, and the effect was significantly greater with microtubule-associated protein (MAP) preparations from embryonic brain than with preparations from the adult. Ser 262 is phosphorylated extensively in paired helical filament (PHF) tau from Alzheimer's disease (AD) brain, to a lesser extent in fetal tau, and only to a very minor extent in biopsy-derived human tau. Because the 100 kDa protein kinase activity phosphorylates Ser 262 and is higher in the fetal brain than the adult brain, it is hypothesized that an inappropriate re-expression and/or re-activation of this or a similar developmentally regulated protein kinase could contribute to the phosphorylation of Ser 262 in PHF-tau, and thus play a role in the pathogenesis of AD.

Amino Acid Sequence↗

PubMed1997

Transglutaminase activity is increased in Alzheimer's disease brain.

Transglutaminase is a calcium-activated enzyme that crosslinks substrate proteins into insoluble, often filamentous aggregates resistant to proteases. Because the neurofibrillary tangles in Alzheimer's disease have similar characteristics, and because tau protein, the major component of these tangles is an excellent substrate of transglutaminase in vitro, transglutaminase activity and levels were measured in control and Alzheimer's disease brain. Frozen prefrontal cortex and cerebellum samples from Alzheimer's disease and control cases matched for age and postmortem interval were used in the analyses. Total transglutaminase activity was significantly higher in the Alzheimer's disease prefrontal cortex compared to control. In addition the levels of tissue transglutaminase, as determined by quantitative immunoblotting, were elevated approximately 3-fold in Alzheimer's disease prefrontal cortex compared to control. To our knowledge, this is the first demonstration that transglutaminase is increased in Alzheimer's disease brain. There were no significant differences in transglutaminase activity or levels in the cerebellum between control and Alzheimer's disease cases. Because the elevation of transglutaminase in the Alzheimer's disease samples occurred in the prefrontal cortex, where neurofibrillary pathology is usually abundant, and not in the cerebellum, which is usually spared in Alzheimer's disease, it can be suggested that transglutaminase could be a contributing factor in neurofibrillary tangle formation.

Aged↗

PubMed1997

Oxidation inhibits substrate proteolysis by calpain I but not autolysis.

In this study, the effects of oxidation on calpain I autolysis and calpain-mediated proteolysis were examined. Calpain I was incubated with increasing concentrations of free calcium in the presence or absence of oxidant, and autolytic conversion of both the 80- and 30-kDa subunits was measured by immunoblotting utilizing monoclonal antibodies which recognize both autolyzed and non-autolyzed forms of each subunit, respectively. Autolytic conversion of the 80-kDa subunit of calpain I was not detected until free calcium concentration was greater than 40 microM, whereas autolysis of the 30-kDa subunit did not occur until the free calcium concentration was greater than 100 microM. In addition, autolytic conversion of either the 80- or 30-kDa subunit was not inhibited by the presence of oxidant. Calpain I activity was measured using the fluorescent peptide N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4- methylcoumarin or the microtubule-associated protein tau as substrate. Calpain I was found to have proteolytic activity at free calcium concentrations below that required for autolysis. Calpain I activity was strongly inhibited by oxidant at all calcium concentrations studied, suggesting that proteolytic activity of both the non-autolyzed 80-kDa and autolyzed 76-kDa forms was susceptible to oxidation. Interestingly, whereas oxidation did not inhibit autolytic conversion, the presence of high substrate concentrations did result in a significant reduction of autolysis without altering calpain proteolytic activity. Calpain I activity that had been inhibited by the presence of oxidant was recovered immediately by addition of the reducing agent dithiothreitol.

Calcium↗

PubMed1997

Calpains: intact and active?

Calpains are a family of calcium-dependent thiol-proteases which are proposed to be involved in many physiological processes as well as pathological conditions. Calpains are likely to be involved in processing of numerous enzymes and cytoskeletal components, thereby linking their activity to a variety of intracellular events. Although widely studied, the precise mechanism(s) involved in calpain activation and activity in vivo remain poorly understood. Initial studies suggested that calpain exists primarily as an inactive proenzyme that required autolytic cleavage for activation. It was also hypothesized that calpain associated with membrane phospholipids, serving to increase calcium sensitivity, facilitating autolytic conversion and thus activating the enzyme. These hypotheses, however, have not been universally accepted and there is increasing evidence that intact, non-autolyzed calpain is the physiologically active calpain form.

Calcium↗

PubMed1997

Large-scale production and characterization of Bacillus thuringiensis subsp. tenebrionis insecticidal protein from Escherichia coli.

Bacillus thuringiensis subsp. tenebrionis insecticidal protein was produced in recombinant Escherichia coli and purified to near homogeneity to provide quantities of protein for safety-assessment studies associated with the registration of transgenic potato plants. The 68-kDa protein is produced naturally by Bacillus thuringiensis subsp. tenebrionis by translation initiation at an internal initiation site in the native DNA sequence. The gene sequence specific for this truncated protein was expressed in E. coli strain JM 101 and fermented at the 1000-1 scale. The protein accumulated as insoluble inclusion bodies, and was purified by extraction at pH 10.8 with carbonate buffer, selective precipitation at pH 9.0, and differential centrifugation. No chromatography steps were required to produce over 50 g purified protein as a lyophilized powder with a purity greater than 95% and demonstrating full insecticidal activity against Colorado potato beetle larvae. The protein was further characterized to assure identity and suitability for use in safety-assessment studies.

Animals↗

PubMed1997

The tau protein in human cerebrospinal fluid in Alzheimer's disease consists of proteolytically derived fragments.

Previous studies have shown that the levels of the microtubule-associated protein tau in the CSF of patients with Alzheimer's disease (AD) are elevated compared with age-matched controls. In spite of these findings, the nature of tau in CSF has not been well documented. In the present study, tau was immunoprecipitated from CSF of patients with AD or acute stroke, as well as normal elderly controls, followed by immunoblot analysis. In all cases, CSF tau consisted primarily of a band migrating at 26-28 kDa. In AD and stroke patients, several smaller tau fragments were also detected. No intact tau was detected in any of the CSF samples examined. Further immunoprecipitation studies showed that the majority of the tau fragments contained the amino terminus of the molecule. Treatment of CSF tau with alkaline phosphatase did not alter the electrophoretic properties of the fragments. These studies clearly demonstrate that CSF tau is truncated rather than intact.

Alzheimer Disease↗

PubMed1997

Select alterations in protein kinases and phosphatases during apoptosis of differentiated PC12 cells.

The involvement of cell cycle-regulatory proteins in apoptosis of neuronally differentiated PC12 cells induced by the removal of nerve growth factor and serum was examined. Three major findings are presented. (1) Cdc2 kinase protein levels increased fivefold in apoptotic PC12 cells by day 3 of serum and nerve growth factor deprivation. Histone H1 kinase activity was increased significantly in p13(suc1) precipitates of apoptotic PC12 cells, which was due to increased activation and/or expression of cdc2 kinase. (2) The protein levels of cyclin-dependent kinase 4, cyclin D, and proliferating cell nuclear antigen that are normally expressed in the cell cycle were increased during neuronal PC12 cell apoptosis. (3) The levels of the catalytic subunit, but not the regulatory subunit of the calcium/calmodulin-dependent protein phosphatase 2B, decreased significantly concomitant with a significant decrease in protein phosphatase 2B activity early in the apoptotic process. Protein phosphatase 2A activity decreased slightly but significantly after 3 days of serum and nerve growth factor deprivation, and no alterations in protein phosphatase 1 were observed during the apoptotic process. These data demonstrate that certain cell cycle-regulatory proteins are inappropriately expressed and that alterations in specific phosphorylation events, as indicated by the increase in histone H1 kinase activity and the decrease in protein phosphatase 2B activity, are most likely occurring during apoptosis of PC12 cells. These observations support the hypothesis that apoptosis may be due in part to a nondividing cell's uncoordinated attempt to reenter and progress through the cell cycle.

Animals↗

PubMed1997

Nerve growth factor protects PC12 cells against peroxynitrite-induced apoptosis via a mechanism dependent on phosphatidylinositol 3-kinase.

Nerve growth factor (NGF) prevents apoptosis induced by the oxidant peroxynitrite in undifferentiated PC12 rat pheochromocytoma cells. Previous studies have shown that activation of phosphatidylinositol 3-kinase (PI 3-kinase) by NGF via the TrkA receptor tyrosine kinase protects PC12 cells from serum deprivation-induced apoptosis. We found that two PI 3-kinase inhibitors, wortmannin and LY294002, eliminated the protection NGF provided against peroxynitrite-induced apoptosis at concentrations consistent with their effectiveness as PI 3-kinase inhibitors. When the activity of PI 3-kinase was assayed in phosphotyrosine immunoprecipitates after treatment of PC12 cells with peroxynitrite, PI 3-kinase activity was reduced by 50% of that detected in control cells, whereas PI 3-kinase activity in NGF-treated cells was unaffected by peroxynitrite. If an antibody against PI 3-kinase was used to immunoprecipitate the enzyme, treatment with peroxynitrite had no effect on activity. Therefore, peroxynitrite appeared to disrupt interactions between PI 3-kinase and phosphotyrosine proteins, rather than directly inhibiting the enzyme. NGF also activates p21Ras-dependent pathways, but this did not appear to be required for NGF to exert its protective effect against peroxynitrite. PC12 cells expressing a dominant inhibitory mutant of p21Ras were equally susceptible to peroxynitrite-induced apoptosis, which was prevented by NGF. Wortmannin was also able to block the protective effect of NGF in the p21Ras mutant cell line. Although many signaling pathways are activated by NGF, these results suggest that a PI 3-kinase-dependent pathway is important for inhibiting peroxynitrite-induced apoptosis.

Animals↗

PubMed1997

Ceramide selectively decreases tau levels in differentiated PC12 cells through modulation of calpain I.

Ceramide has been recently proposed to be a signal mediator in several important physiological processes including apoptosis, cellular growth, and differentiation. Because the microtubule-associated protein tau plays an important role in the establishment and maintenance of neuronal morphology, the effects of ceramide on tau were examined. Treatment of differentiated PC12 cells with the cell-permeable ceramide derivative N-acetylsphingosine (C2) resulted in a significant reduction in tau levels. Significant decreases in tau levels were also observed when the cells were treated with another ceramide derivative, N-hexanoylsphingosine (C6). In addition, C2 treatment increased the levels of a calpain-derived spectrin breakdown product but did not alter the levels of two cytoskeletal proteins, alpha-actin and alpha-tubulin. Because both tau and spectrin are proteolyzed in vitro by the calcium-activated cysteine protease calpain, the effects of ceramide analogues on the activity of this protease were examined. Treatment of PC12 cells with C2 enhanced calcium-stimulated proteolytic activity significantly, as revealed by monitoring the hydrolysis of the membrane-permeable calpain-selective fluorescence probe N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4-methylcoumarin . This activity increase was not due to a direct effect of C2 on calpains, because C2 did not alter the activities of purified calpain I or II. In addition, C2 treatment of PC12 cells resulted in a significant increase in the levels of calpain I and, to a lesser extent, the levels of calpastatin (an endogenous calpain inhibitor protein), whereas the levels of calpain II were not changed. Moreover, treatment of the cells with the synthetic calpain-specific inhibitor N-carbobenzoxy-L-leucyl-L-leucyl-L-tyrosine diazomethyl ketone blocked the C2-induced decreases in tau levels. These results indicate that tau levels are regulated in response to a physiological factor and, thus, have implications for ceramide-mediated changes in normal and pathological neuronal processes.

Actins↗

PubMed1996

The microtubule-associated protein tau is extensively modified with O-linked N-acetylglucosamine.

Tau is a family of phosphoproteins that are important in modulating microtubule stability in neurons. In Alzheimer's disease tau is abnormally hyperphosphorylated, no longer binds microtubules, and self-assembles to form paired helical filaments that likely contribute to neuron death. Here we demonstrate that normal bovine tau is multiply modified by Ser(Thr)-O-linked N-acetylglucosamine, a dynamic and abundant post-translational modification that is often reciprocal to Ser(Thr)-phosphorylation. O-GlcNAcylation of tau was demonstrated by blotting with succinylated wheat germ agglutinin and by probing with bovine milk beta(1,4)galactosyltransferase. Structural analyses confirm the linkage and the saccharide structure. Tau splicing variants are multiply O-GlcNAcylated at similar sites, with an average stoichiometry of greater than 4 mol of O-linked N-acetylglucosamine/mol of tau. However, the number of sites occupied appears to be greater than 12, suggesting substoichiometric occupancy at any given site. A similar relationship between average stoichiometry and site-occupancy has also been described for the phosphorylation of tau. Site-specific or stoichiometric changes in O-GlcNAcylation may not only modulate tau function but may also play a role in the formation of paired helical filaments.

Acetylglucosamine↗

PubMed1996

Tau protein is phosphorylated by cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II within its microtubule-binding domains at Ser-262 and Ser-356.

Phosphorylation of tau protein at Ser-262 has been shown to diminish its ability to bind to taxol-stabilized microtubules. The paired helical filaments (PHFs) found in Alzheimer's disease brain are composed of PHF-tau, which is hyperphosphorylated at multiple sites including Ser-262. However, protein kinase(s) able to phosphorylate this site are still under investigation. In this study, the ability of cyclic AMP-dependent protein kinase (cAMP-PK) and calcium/calmodulin-dependent protein kinase II (CaMKII) to phosphorylate tau at Ser-262, as well as Ser-356, is demonstrated by use of a monoclonal antibody (12E8) which has been shown to recognize tau when these sites are phosphorylated. Cleavage of cAMP-PK-phosphorylated tau at cysteine residues by 2-nitro-5-thiocyanobenzoic acid, which cuts the protein into essentially two fragments and separates Ser-262 from Ser-356, revealed that cAMP-PK phosphorylates both Ser-262 and Ser-356. In addition, phosphorylation with cAMP-PK or CaMKII of recombinant tau in which Ser-262, Ser-356 or both had been mutated to alanines, clearly demonstrated that cAMP-PK and CaMKII were able to phosphorylate both sites. Mitogen-activated protein kinase or protein kinase C did not phosphorylate tau at Ser-262 and/or Ser-356. Finally, evidence is presented that phosphorylation of both these sites occurs in cultured nerve cells under certain conditions, indicating their potential physiological relevance.

Amino Acid Sequence↗