Modulation of the in Situ Activity of Tissue Transglutaminase by Calcium and GTP*

Jianwen Zhang, Mathieu Lesort, Rodney P. Guttmann, and Gail V. W. JohnsonDagger

From the Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0017

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Tissue transglutaminase (tTG)1 is a novel, dual function protein having both transamidating activity and a role as a signal-transducing GTP-binding protein (1, 2). As a member of the transglutaminase family, tTG catalyzes a calcium-dependent acyl transfer reaction between the gamma -carboxamide of a peptide-bound glutamine residue and the epsilon -amino group of a peptide-bound lysine, or the primary amino group of a polyamine, yielding either an isopeptide bond or a (gamma -glutamyl)polyamine bond, respectively (1). This transamidating activity of tTG is inhibited by GTP, an effect that is reversed by an intrinsic GTPase activity of tTG (3, 4). GTP-bound tTG was subsequently shown to function as a signal-transducing GTP-binding protein (Galpha h), which couples activated receptors to phospholipase Cdelta , resulting in stimulation of this effector enzyme (2, 5). Thus, this intriguing protein can serve the cell in two apparently unrelated capacities, its role apparently determined by incompletely characterized intracellular regulators.

tTG is found in many different mammalian cells and tissues and has been implicated as a participant in a vast array of physiological and pathological processes. In its capacity as a transamidating enzyme, tTG has been proposed to play an important role in bone development (6), axonal growth and regeneration (7, 8), modulation of cell adhesion (9, 10), differentiation and apoptosis (11, 12), and tumor growth and metastasis (13, 14). Recent studies have begun to elucidate the specific roles of tTG in these different biochemical processes. For example, tTG is likely to be involved in the activation of both midkine, a heparin-binding growth/differentiation factor, and interleukin 2 by catalyzing the formation of stable dimers (7, 8, 15). It has also been suggested that tTG contributes to the transforming growth factor-beta activation process by cross-linking the large latent transforming growth factor-beta complex to the extracellular matrix (16). Additionally, there are data to indicate that tTG is involved in stabilizing tissue during wound healing by cross-linking anchoring fibrils and, more specifically, collagen VII (17).

In addition to catalyzing the formation of isodipeptide bonds, tTG in its role as a transamidating enzyme covalently incorporates polyamines into substrate proteins. Protein-polyamine conjugates have been detected in several tissues and cell lines (18, 19), and in vitro tTG incorporates polyamines into numerous proteins (20-22). Although functional changes resulting from tTG-catalyzed incorporation of polyamines into proteins have not been well defined, previous studies have shown that in vitro the covalent incorporation of polyamines into phospholipase A2 increases activity (23).

In its capacity as a signal transducing GTP-binding protein tTG has been designated Galpha h, a protein that forms noncovalent heterodimers with a 50-kDa protein (2, 24). Galpha h has been shown to couple to alpha 1-adrenoreceptors and thereby mediate the activation of phospholipase Cdelta (2, 5). Recent data indicate that in the heart the alpha 1-adrenoreceptor couples to Galpha h, and in cardiomyopathic heart tissue the intrinsic activity of Galpha h is decreased (25). The reason for the decrease in Galpha h activity and GTP binding in the failing heart is unknown; however, it has been suggested that other proteins, such as the 50-kDa protein that binds Galpha h in a GTP-dependent manner, may be involved (24).

Because tTG is apparently involved in multiple cellular processes, its expression and activation are likely to be tightly regulated processes. Interleukin 6 has been shown to induce tTG expression in hepatocytes (26), and cAMP induces expression in cerebellar granule cells (27). In many, but not all, cell types, retinoids are effective inducers of tTG expression (28-31). Nagy et al. (32) have demonstrated that the mouse tTG promoter is activated by retinoid activation of either retinoic acid receptor-retinoid X receptor heterodimers or retinoid X receptor homodimers. Retinoic acid receptor and retinoid X receptor are highly regulated and exhibit specific temporal, spatial, and tissue-specific expression patterns (33) and therefore are likely to be important determinants in tTG expression. The in vitro regulation of tTG transglutaminating activity by calcium and GTP has been well documented (4, 34, 35). However, the in situ modulation of tTG by these and other factors has not been thoroughly examined. Therefore, the purpose of this study was to examine the modulation of tTG by calcium and GTP in situ.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Chemicals-- N,N-Dimethylated casein, bovine serum albumin (BSA), retinoic acid (RA), ionomycin, carbachol, putrescine dihydrochloride, Tween 20, Bisbenzamide (Hoescht), poly-D-lysine (30,000-70,000 daltons), and o-phenylenediamine dihydrochloride were purchased from Sigma; thapsigargin was from Alexis; phenylmethylsulfonyl fluoride, SDS, and FITC-conjugated streptavidin were purchased from Boehringer Mannheim; [1,4-3H]putrescine dihydrochloride (1 mCi/ml) and the enhanced chemiluminescence (ECL) reagents were purchased from Amersham Corp.; 5-(biotinamido)pentylamine, horseradish peroxidase (HRP)-conjugated streptavidin, and BCA protein assay reagents were purchased from Pierce; and N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-tyrosine diazomethyl ketone (Cbz-LLY-DMK) was from Molecular Probes. Calpain I, Nonidet P-40, and Bay K8644 were purchased from Calbiochem, and Tiazofurin was a gift from NCI, National Institutes of Health. RPMI 1640 was purchased from Cellgro; penicillin/streptomycin and horse serum were from Life Technologies, Inc.; and fetal clone II was purchased from Hyclone. The tTG monoclonal antibody 4C1 was produced by the Hybridoma Core Facility at the University of Alabama at Birmingham (36); the monoclonal antibody to type I TG was from Biomedical Technologies; the monoclonal tTG antibody CUB 7402 was from Neomarkers; and HRP-conjugated goat anti-mouse IgG was purchased from Bio-Rad. Texas red-conjugated goat anti-mouse IgG was from Jackson ImmunoResearch. Fura-2 was from TefLabs, and N-succinyl-L-leucyl-L-leucyl-L-tyrosine-7-amido-4-methylcoumarin was from Bachem. For these experiments, maitotoxin (MTX) was purchased from Calbiochem. However, it should be noted that the potency of MTX varies significantly depending on the vendor; therefore, initial experiments to determine the effective doses of the drug should be carried out. All other reagents were from Sigma.

Cell Culture-- Human neuroblastoma SH-SY5Y cells were maintained on Corning dishes in RPMI 1640 medium supplemented with 20 mM glutamine, 10 units/ml penicillin, 100 µg/ml streptomycin, 5% fetal clone II serum, and 10% horse serum. For differentiation, the percentage of fetal clone II serum and horse serum in the media were reduced to 1 and 4%, respectively. To initiate differentiation, cells were grown in the low serum medium containing 20 µM RA. The differentiating medium supplemented with 20 µM RA was replaced every 48 h. All experiments were carried out on subconfluent cultures.

Immunoblotting-- To evaluate the expression level of tTG in cells during differentiation, extracts from cells were prepared and quantitatively immunoblotted. Cells were harvested in cold phosphate-buffered saline (PBS), collected by centrifugation, resuspended in a homogenizing buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and a 10 µg/ml concentration each of aprotinin, leupeptin, and pepstatin) and sonicated on ice. Protein concentrations of the homogenates were determined using the BCA method and diluted to a final concentration of 1 mg/ml with 2 × reducing stop buffer (0.25 M Tris-HCl, pH 6.8, 5 mM EDTA, 5 mM EGTA, 25 mM dithiothreitol, 2% SDS, and 10% glycerol with bromphenol blue as the tracking dye). Samples (25 µg of protein) were resolved on 8% SDS-polyacrylamide gels and transferred to nitrocellulose. Blots were blocked in 5% nonfat dry milk in TBST (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.05% Tween 20) for 1 h at room temperature. The blots were then incubated with 1-2 µg/ml anti-tTG monoclonal antibody 4C1 in the same buffer for 2 h at room temperature. The blots were rinsed once with TBST and incubated with HRP-conjugated goat anti-mouse IgG (1:3000) in TBST for 1 h at room temperature. The blots were rinsed three times for 30 min with TBST, followed by four quick rinses with distilled water, and developed with ECL. The immunoblots were analyzed using a Bio-Rad GS-670 imaging densitometer and normalized to an internal standard to eliminate blot to blot variation in staining intensity. Data were expressed as a percentage of the maximal level of tTG ± S.E.

In Vitro Putrescine Incorporation Assay-- In vitro tTG activity was measured in cell extracts using a modification of the procedure of Hand et al. (11) as described previously (36).

In Situ tTG Activity Assay-- For in situ tTG activity measurements, SH-SY5Y cells were preincubated with 5-(biotinamido)pentylamine, a biotinylated polyamine, and incorporation of the reagent into proteins was determined (27). Prior to treatment with the indicated drugs or appropriate vehicles (controls), cells were incubated for 1 h with 2 mM 5-(biotinamido)pentylamine which was prepared as a 100 mM stock in 50 mM Tris-HCl, 150 mM NaCl, pH 7.5. MTX, carbachol, and tiazofurin were dissolved in water; ionomycin and thapsigargin were dissolved in Me2SO; and Bay K8644 was dissolved in ethanol. The maximal Me2SO or ethanol concentration to which the cells were exposed was 0.1%. Cells were treated with the drugs as indicated and then harvested and lysed as described above. Ten micrograms of homogenate protein was diluted to 50 µl with coating buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EGTA, 5 mM EDTA) and added to each well of a 96-well microtiter plate (Falcon), and the plates were incubated overnight at 4 °C. Two hundred microliters of 5% BSA, 0.01% SDS, 0.01% Tween 20 in borate-buffered saline (BBS; 100 mM boric acid, 20 mM sodium borate, 80 mM NaCl) was added to each well, and the incubation continued for 2 h at 37 °C. The mixture was removed, and each well was rinsed once with 1% BSA, 0.01% Tween 20 in BBS. One hundred microliters of HRP-conjugated streptavidin (1:1000) in 1% BSA, 0.01% Tween 20 was added to each well and incubated at room temperature for 1 h. The wells were rinsed four times with 1% BSA, 0.01% Tween 20 in BBS, and then 200 µl of substrate solution (0.4 mg of o-phenylenediamine dihydrochloride/ml of 0.05 M sodium citrate phosphate buffer, pH 5.0) was added to each well. After incubating 10-20 min at room temperature, the reactions were stopped by the addition of 50 µl of 3 N HCl to each well, and the presence of proteins into which 5-(biotinamido)pentylamine had been incorporated was quantitated by measuring the absorbance at 492 nm on a microplate spectrophotometer (Molecular Devices). All measurements were done in triplicate and repeated at least three times. The activity of tTG in situ was calculated as a percentage of basal activity (i.e. no drug additions) within a given group of samples. Data were analyzed using Student's t test, and values were considered significantly different when p < 0.05. To visualize the proteins into which the 5-(biotinamido)pentylamine had been incorporated, 2 µg of the homogenates was resolved on a 8% polyacrylamide SDS gel; transferred to nitrocellulose; blocked in 5% BSA, 0.05% SDS, 0.01% Nonidet P-40 in BBS for at least 2 h at room temperature; and incubated with HRP-conjugated streptavidin (1:2000) in 1% BSA, 0.05% SDS, 0.01% Nonidet P-40 in BBS for 2 h at room temperature. The blots were rinsed three times for 30 min each with the same buffer followed by four quick rinses with distilled water. The blots were then developed following the standard ECL protocol. Except where indicated, studies were carried out on cells that had been treated with RA for 6 days.

Cytochemistry-- For cytochemical analysis, SH-SY5Y cells were replated onto poly-D-lysine-coated coverslips in 24-well plates. Cells were preincubated for 1 h at 37 °C in the presence of 2 mM 5-(biotinamido)pentylamine and subsequently treated with 1 nM MTX for 20 min. Cells not exposed to MTX served as controls. After treatment, SH-SY5Y cells were fixed in paraformaldehyde (4% in PBS) for 30 min at room temperature, incubated for 1.5 min with 0.2% Triton X-100 in PBS, and rinsed three times for 5 min each with PBS, prior to incubation with 3% BSA in PBS for 30 min to reduce the background. Cells were then incubated for 1 h with the tTG monoclonal antibody CUB 7402 (diluted 1:10 in PBS containing 0.1% BSA), rinsed three times for 5 min each with PBS, and incubated for 1 h at room temperature with FITC-conjugated streptavidin diluted 1:100 in PBS and Texas red-conjugated goat anti-mouse IgG diluted 1:50 in PBS. Cells were counterstained with the nuclear dye Hoescht (5 µg/ml) for the evaluation of the cell number, and coverslips were washed extensively in PBS prior to mounting. Controls contained either no primary antibody or no 5-(biotinamido)pentylamine. Cells were viewed with a Nikon Diaphot 300 epifluorescence microscope, and images were captured with a Photometric CH250 CCD camera; digitally stored images were combined and displayed with the IP Lab Spectrum software.

GTP Measurements-- The levels of GTP in the cells were determined using reverse phase HPLC as described previously (37). In brief, cells were rinsed once with PBS and collected into 1 M formic acid. The samples were vortexed and spun at 16,000 × g for 4 min at 4 °C. The supernatants were lyophilized, and the pellets were used for protein determinations. The lyophilized samples were resuspended in HPLC grade water, injected onto a C-18 column (Vydac, 250 × 4.6 mm, 5 µm), and eluted with a 4-42% acetonitrile gradient. Nucleotide standards were used to determine the position at which GTP eluted. All data were normalized to mg of protein in each sample.

Intracellular Calcium Levels-- Intracellular calcium levels were measured in cultured cells using Fura-2 essentially as described previously (38, 39) with a few modifications. In brief, cells were grown on coverslips, and prior to experimentation they were loaded with a 5 µM concentration of the acetoxymethyl ester form of Fura-2. Coverslips were placed in an imaging chamber (Warner Instrument Co.) and mounted in a heater platform on the stage of a Nikon Diaphot. The cells were maintained at 37 °C in a Ringer's solution for the duration of the experiments. Images were obtained using an Ionoptix ICCD camera (Ionoptix Corp., Milton, MA) and processed with the IonWizard software program. Intracellular calcium concentrations were determined from the ratio of fluorescence using excitation wavelengths of 340 and 380 nm. Background fluorescence was taken from regions not containing cells and subtracted from the cell images at each wavelength. The ratio of fluorescence in the digitized images is a direct indicator of intracellular calcium concentrations. The system was calibrated following standardized protocols (40).

In Situ Calpain Activity-- Calpain activity was measured in SH-SY5Y using the membrane-permeable, calpain-selective fluorescent peptide, N-succinyl-L-leucyl-L-leucyl-L-tyrosine-7-amido-4-methylcoumarin as described previously (41).

Cell Viability-- The release of the intracellular enzyme lactate dehydrogenase (LDH) into the media was used as a quantitative measure of cell viability. The measurement of LDH was carried out as described previously (42). The percentage of LDH released was defined by LDH activity in the media divided by total LDH activity.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Effects of RA on tTG Expression and in Vitro TG Activity-- Previously, it had been demonstrated that treatment of human neuroblastoma SK-N-BE cells with RA increased tTG gene expression and tTG protein levels (29). To determine if this was also the case for SH-SY5Y human neuroblastoma cells, the cells were treated with 20 µM RA, and the level of tTG expression was determined at various times for 12 days. The levels of tTG increased rapidly after RA treatment. In Fig. 1, a representative immunoblot shows that prior to RA treatment the levels of tTG are virtually undetectable, but after RA treatment tTG levels increase rapidly for 3 days and remain elevated for 12 days. A quantitation of the increase in tTG levels in RA-treated SH-SY5Y cells is shown in Fig. 2. Type 1 (keratinocyte) TG was not detected in naive or RA-treated cells (data not shown). In vitro measurements of TG activity were carried out to determine if the increased levels of tTG correlated with increased activity (Fig. 2). RA treatment of SH-SY5Y cells also resulted in a rapid increase in in vitro TG activity that correlated (r2 = 0.959) with the increased expression levels of tTG. The TG activity in cells treated with RA for 9 days was approximately 10-fold higher than the activity measured in untreated cells.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1.   Representative immunoblot of the induction of tTG in response to RA treatment. SH-SY5Y cells were treated with 20 µM RA for the times indicated, and immunoblots were probed with the monoclonal tTG antibody 4C1.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   Quantitative analysis of the effects of RA treatment on the expression of tTG (bullet ) and in vitro TG activity (black-triangle). Treatment of SH-SY5Y cells with RA for the times indicated resulted in a concomitant increase in both the expression of tTG and in vitro TG activity. The graph is logarithmic.

In Situ TG Activity-- To determine if basal in situ TG activity also correlated with the increases in tTG expression, 5-(biotinamido)pentylamine was used as a probe for endogenous tTG activity. TGs react with free glutamine residues in substrate proteins releasing ammonia, and then the enzyme-substrate intermediate reacts with an appropriate nearby primary amine. This can be either the epsilon -amino group of lysine in an adjacent protein, resulting in an epsilon -(gamma -glutamyl)lysine isodipeptide bond, or the primary amino group of a polyamine, resulting in the covalent incorporation of the polyamine into the protein by a (gamma -glutamyl)polyamine bond (1). Therefore, cells were incubated with 5-(biotinamido)pentylamine, and incorporation of this polyamine derivative into proteins was used as a measure of in situ TG activity (27). Although in vitro TG activity increased concurrently with the RA-induced increases in tTG levels and was significantly elevated at day 1, basal in situ activity was not significantly increased until day 6 of RA treatment, indicating that basal in situ TG activity is tightly regulated by endogenous control mechanisms (Fig. 3).


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3.   Representative blot (A) and quantitative analysis (B) of the TG-catalyzed incorporation of 5-(biotinamido)pentylamine into proteins as a measure of in situ activity. SH-SY5Y cells were treated with RA for the number of days indicated, labeled with 5-(biotinamido)pentylamine, and incubated for 20 min in the absence (-) or presence (+) of 5 nM MTX. A, blots probed with HRP-conjugated streptavidin indicate that basal (-) TG activity increased only slightly in response to RA treatment, while MTX-stimulated (+) TG activity was increased significantly in response to elevated tTG levels (Fig. 1) as a result of RA treatment. Arrows at the left indicate positions at which molecular mass standards (kDa) migrated. B, quantitative analysis of the TG-catalyzed incorporation of 5-(biotinamido)pentylamine into proteins using a microplate-based assay demonstrated that induction of tTG in response to RA treatment resulted in significant increases in MTX-stimulated (+) TG activity. In contrast, basal activity (-) was elevated to a significantly lesser extent. Results were considered significantly different when p was <0.05 (n = 3 separate experiments, each in triplicate).

Because tTG is a calcium-activated enzyme (1), intracellular levels of calcium were elevated by treating the cells with MTX, and endogenous TG activity was measured. MTX has been shown to activate both voltage-sensitive and ligand-gated calcium channels (43) and stimulate inositol phosphate production in a dose-dependent manner (44). In contrast to basal TG activity, the calcium-stimulated increases in in situ TG activity correlated with the observed increases in tTG levels (r2 = 0.758) after RA treatment (Fig. 3).

The Regulation of in Situ TG Activity by Calcium and GTP-- To further assess the effects of increasing intracellular calcium concentrations on TG activity, SH-SY5Y cells were treated with RA for 6 days, preincubated with 5-(biotinamido)pentylamine, and then challenged for 20 min with concentrations of MTX ranging from 0.25 to 40 nM (Fig. 4A). A significant increase in in situ TG activity occurred with 1 nM MTX compared with untreated cells. This finding correlates well with previous data indicating that 1 nM MTX significantly elevates intracellular calcium levels in SH-SY5Y cells (43). No loss of cell viability as determined by an increase in LDH release was observed with MTX doses of 5 nM or less. Intracellular calcium concentrations increased approximately 10-fold in response to 5 nM MTX from approximately 70 nM to 700 nM. Although maximal activation of TG activity occurred at concentrations greater than 5 nM MTX, these higher doses of MTX result in detachment of the cells from the dishes and decreased viability; therefore, 5 nM MTX was used in further investigations. Using 5 nM MTX, the time course of the calcium activation of endogenous TG activity was determined (Fig. 4B). Between 5 and 20 min, there was a rapid increase in TG activity, which reached a plateau after 40 min of incubation with MTX (Fig. 4B); in the same treatment paradigm, maximal intracellular calcium levels occurred by 10 min and were maintained for the duration of the experiment.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4.   Dose- (A) and time-dependent (B) increases in in situ TG activity in response to MTX, which increases intracellular calcium levels. The time-dependent studies in B were carried out with 5 nM MTX. SH-SY5Y cells were treated with 20 µM RA for 6 days prior to use.

Because the transamidating activity of tTG has been shown to be inhibited by GTP in vitro (4), the effects of decreasing GTP levels in the cell on endogenous TG activity was determined. To accomplish this, tiazofurin, a C-nucleoside that selectively and potently inhibits IMP dehydrogenase and decreases GTP levels (45), was used. Treatment of SH-SY5Y cells with concentrations of tiazofurin up to 50 µM resulted in a concentration-dependent increase in in situ TG activity (Fig. 5A). Treatment of SH-SY5Y cells with 50 µM tiazofurin decreased intracellular GTP levels to 22 ± 3% of control values with no loss of cell viability or increased intracellular calcium levels. However, at concentrations of tiazofurin greater than 50 µM, no further increases in in situ TG activity were observed, and activity levels declined at the highest concentrations of tiazofurin that were used (Fig. 5A). A contributing factor to this decline in in situ TG activity is likely to be the fact that at the higher concentrations of tiazofurin there is cellular toxicity. At the highest concentration of tiazofurin (800 µM), the release of LDH was almost 3-fold greater than that of the untreated cells (17 ± 2% for the treated cells compared with 6 ± 1% for the controls). Therefore, the concentration of tiazofurin used to examine the time course of the GTP-depleting effects on in situ TG activity was 50 µM. These studies revealed that maximal activation of TG occurred after 40 min of incubation with tiazofurin (Fig. 5B).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.   Dose- (A) and time-dependent (B) changes in in situ TG activity in response to tiazofurin, which inhibits IMP dehydrogenase and decreases intracellular GTP levels. The time-dependent studies in B were carried out with 50 µM tiazofurin. SH-SY5Y cells were treated with 20 µM RA for 6 days prior to use.

Cellular Localization and Activity of tTG-- To assess the localization of tTG as well as TG activity, a cytochemical approach was used. SH-SY5Y cells that had been treated with RA for 6 days were incubated with 5-(biotinamido)pentylamine and then treated with 1 nM MTX for 20 min. Control and MTX-treated cells were fixed and immunostained with a monoclonal antibody to tTG and were also probed with FITC-conjugated streptavidin to localize polyamine-modified proteins to obtain a measure of endogenous TG activity. Treatment of SH-SY5Y cells with RA resulted in a significant increase in the expression of tTG in most cells (Table I; Fig. 6, A and D), and the unstimulated cells exhibited little basal TG activity (Table I; Fig. 6, B and C). Treatment of cells within 1 nM MTX results in a significant increase in TG activity in almost all of the cells that express tTG (Table I; Fig. 6, D-F). Some TG activity was observed in the nucleus; however, this was not unexpected, since previous studies have demonstrated that tTG is found in the nucleus (46). Interestingly, transamidating activity appeared to be perinuclear and therefore also proximal to the endoplasmic reticulum (ER) (Fig. 6, E and F).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Percentage of SH-SY5Y cells that are tTG-positive and show TG activity either in the absence (-MTX) or presence (+MTX) of maitotoxin
Undifferentiated SH-SY5Y cells were maintained in the absence of RA, while differentiated cells were treated with 20 µM RA for 6 days prior to use. Cells were incubated in the absence or presence of 1 nM MTX, fixed, and stained as described under "Experimental Procedures." The percentage of cells that were tTG-positive and/or showed cytosolic TG activity was determined for each field. For each condition, at least 15 separate fields from two individual experiments were used for the quantitation, and a minimum of 200 cells were counted.


View larger version (71K):
[in this window]
[in a new window]
 
Fig. 6.   Cytochemical analysis of tTG distribution and activity in SH-SY5Y cells treated with 20 µM RA for 6 days. Cells were labeled with 5-(biotinamido)pentylamine and then incubated in the absence (A-C) or presence (D-F) of 1 nM MTX for 20 min prior to fixation and staining. All cells were double-labeled with tTG antibody (tTG localization) and FITC-streptavidin (TG activity) and stained with Hoescht. In all panels, tTG is in red, TG activity (as indicated by FITC-streptavidin staining) is in green, and the nuclei are in blue (Hoescht staining). Panels A and B show tTG staining (A) and TG activity (B) of the same cells, and panels D (tTG staining) and E (TG activity) are from the same field of cells. In unstimulated (no MTX) cells (panels A and B), tTG is expressed in most cells (A), and TG activity is very low (B). In MTX-treated cells (panels D and E), tTG expression is also evident (D); however, significant levels of TG activity were detected (E). The original magnification of the images shown in panels A, B, D, and E was × 400. To analyze the colocalization of tTG and TG activity, composite images of tTG (red), TG activity (green), and Hoescht staining (blue) were created at an original magnification of × 1000. In panel C, a composite image of untreated SH-SY5Y cells shows only the presence of tTG; no TG activity is evident. In panel F, a composite image of MTX-treated SH-SY5Y cells shows TG activity and the colocalization of tTG expression and activity (red + green = yellow/orange). The proximity of TG activity to the nucleus is also evident in the image shown in panel F.

Interaction of the Effects of Calcium and GTP Depletion on Endogenous TG Activity-- To determine how increased intracellular calcium concentrations and decreased GTP concentrations cooperatively modulate endogenous TG activity, experiments in which cells were treated with both MTX and tiazofurin were carried out. Individually, both MTX and tiazofurin increase TG activity. However, the endogenous TG activity in the cells treated with both MTX and tiazofurin was less than that in cells treated with MTX alone (Fig. 7A). Previous studies demonstrated that in vitro GTP inhibits the proteolysis of tTG by trypsin (4). Considering that MTX activates the calcium-dependent protease calpain in SH-SY5Y cells (43), the effects of the selective membrane-permeable calpain inhibitor Cbz-LLY-DMK (47) on the MTX and tiazofurin-induced activity of TG was examined. The presence of 15 µM Cbz-LLY-DMK blocked the decrease in TG activity elicited by MTX in the presence of tiazofurin resulting in an almost additive increase in activity (Fig. 7A). Immunoblot analysis of the levels of tTG in these cells clearly demonstrated that the combination treatment of MTX and tiazofurin resulted in a significant decrease in tTG levels and that tTG degradation was inhibited by the calpain inhibitor Cbz-LLY-DMK (Fig. 7B). Similar results were obtained with another calpain inhibitor, the peptide aldehyde calpeptin (data not shown). Measurement of in situ calpain activity with a membrane-permeable, calpain-selective fluorescent peptide indicated that treatment with MTX resulted in a significant increase in calpain activity and that this activity was almost completely blocked by the addition of Cbz-LLY-DMK or calpeptin. Tiazofurin alone had no effect on the activity of calpain, nor did it increase MTX-stimulated calpain activity (data not shown). These results suggest that tTG is an endogenous substrate of calpain and that decreasing GTP levels results in increased degradation of tTG in a calcium-dependent manner.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 7.   Modulation of endogenous TG activity (A) and tTG levels (B) by calcium, GTP, and calpain. RA-treated SH-SY5Y cells were treated with 50 µM tiazofurin (TIA) for 40 min, 5 nM MTX for 20 min, or with MTX added for the last 20 min of the 40-min incubation with tiazofurin. In some experiments, 15 µM Cbz-LLY-DMK (CBZ), a membrane-permeable calpain inhibitor, was added at the same time as the tiazofurin. A, individually, both MTX and tiazofurin significantly increased in situ endogenous TG activity; however, tiazofurin and MTX in combination resulted in a significant decrease in TG activity compared with MTX alone. Inhibition of calpain by Cbz-LLY-DMK blocked the decrease in TG activity. Results were considered significantly different when p was <0.05 (n = 3-5 separate experiments, each in triplicate). B, tTG immunoblot, demonstrating that treatment of cells with MTX and tiazofurin results in a loss of tTG and that inhibition of calpain inhibits the degradation of tTG.

Effects of Different Calcium-mobilizing Agents on Endogenous TG Activity-- SH-SY5Y cells that had been treated with RA for 6 days were incubated for 60 min with 10 µM ionomycin (a calcium ionophore), 20 µM Bay K8644 (an L-type calcium channel agonist), 20 µM thapsigargin (an inhibitor of calcium uptake by the ER), 1 mM carbachol (a muscarinic cholinergic receptor agonist), a combination of thapsigargin and carbachol, or no additions. Previous studies have demonstrated that SH-SY5Y cells express L-type calcium channels (48) and M3 receptors that when activated stimulate phospholipase C and therefore production of inositol phosphates (i.e. inositol 1,4,5-trisphosphate) (49). Ionomycin increased significantly endogenous TG activity, whereas Bay K8644 had no effect on TG activity (Fig. 8). Thapsigargin or carbachol alone resulted in a significant increase in TG activity, and treatment of SH-SY5Y cells with carbachol in combination with thapsigargin resulted in an increase in TG activity that was significantly greater than the activity observed with either treatment alone (Fig. 8). The combination of carbachol and thapsigargin resulted in an approximately 3-fold increase in intracellular calcium concentrations. The combined treatment of carbachol and thapsigargin elevated intracellular calcium concentrations approximately 3-fold to 200 nM. These studies reveal that TG can be activated in situ through a receptor-mediated pathway.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 8.   Quantitative analysis of the effects of different calcium-mobilizing agents on in situ TG activity in RA-treated SH-SY5Y cells. Cells were treated for 60 min with 10 µM ionomycin (ION), 20 µM Bay K8644 (BAY), 20 µM thapsigargin (THP), 1 mM carbachol (CRB), a combination of carbachol and thapsigargin, or no drug (CTL). Bay K8644 did not increase TG activity. Ionomycin, thapsigargin, and carbachol all significantly elevated in situ TG activity. Thapsigargin treatment of carbachol-stimulated cells resulted in an increase in TG activity that was greater than the activity observed in the presence of carbachol alone. Results were considered significantly different when p was <0.05 (n = 3 separate experiments, each in triplicate).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The primary goal of this study was to examine the endogenous regulation of TG activity. tTG has been suggested to participate in numerous cellular functions including apoptosis (12, 50), cell cycle progression (51), cell adhesion (9, 10), axonal growth and regeneration (7, 8), and signal transduction (2). Considering this diverse array of cellular processes with which tTG has been associated, it is likely that tTG activity is regulated by specific mechanisms. Previous in vitro studies have demonstrated that tTG is a calcium-dependent enzyme (1) and that GTP inhibits the transamidating activity of tTG (4). Although the modulation of tTG expression in situ has been examined (32), little is known about how calcium and GTP, as well as other factors, modulate the in situ activity of tTG. A previous study utilized electropermeabilization to examine the regulation of TG in ECV-304 human umbilical vein cloned endothelial cells by exogenously added calcium and nucleotides (52). However, endogenous mechanisms regulating TG activity in the cell were not examined in this study. In the present study, calcium and GTP were found to regulate TG activity in situ, both directly and indirectly. Additionally, the activity of TG was demonstrated to be under the influence of a receptor-activated pathway, most likely due to inositol 1,4,5-trisphosphate-stimulated release of calcium from the ER.

To quantitatively measure endogenous TG activity, a modification of preexisting protocols was used. There is uptake of polyamines and derivatives by most cells (53, 54), and previous studies have used 5-(biotinamido)pentylamine to label cells and visualize TG-modified proteins by blotting with HRP-conjugated streptavidin (27). In addition, a microplate in vitro TG assay using 5-(biotinamido)pentylamine and dimethylated casein as the substrate was described previously (55), as well as a procedure to measure incorporation of polyamines into proteins (52). By combining and modifying these approaches, a method to quantitatively measure TG activity in situ was developed (see "Experimental Procedures"). This assay allowed quantitative measurements of in situ TG activity under basal conditions and in response to various stimuli, which permitted differential assessments of total TG activity measured in vitro and actual in situ TG activity. The novelty of this approach is that TG activity was measured in intact cells rather than in cell lysates or permeabilized cells, and therefore evaluation of endogenous regulatory mechanisms was possible.

RA treatment of SH-SY5Y cells enhanced significantly the levels of tTG and the in vitro TG activity. SH-SY5Y is a serially isolated, nonhomogenous neuroblast clonal cell line derived from the parental neuroblastoma cell line SK-N-SH (56, 57). The levels of tTG increased significantly in response to RA treatment as detected by immunoblot analysis (Fig. 1), and tTG was detected in most cells after RA treatment (Table I; Fig. 6, A and D). Additionally, almost all of the SH-SY5Y cells that expressed tTG showed an increase in transamidating activity in response to MTX challenge (Table I; Fig. 6, D-F). The hypothesis that endogenous tTG is tightly regulated is supported by the finding that although tTG levels and in vitro TG activity increase dramatically and rapidly (by day 1) in response to RA treatment, basal in situ activity did not increase until day 6, and the response was less than robust. In contrast, increasing intracellular calcium levels by treatment with MTX increased significantly in situ TG activity, and this increase in activity correlated significantly with the expression levels of tTG.

Previous in vitro studies have demonstrated that tTG possesses intrinsic GTPase activity (3), that GTP inhibits the transamidating activity of tTG (4), and that GTP is required for tTG to act as a signal-transducing G-protein (Galpha h) and activate phospholipase Cdelta (5). However, little is known about how GTP regulates tTG function in vivo. To assess the in situ regulatory function of GTP in modulating tTG activity, tiazofurin, a selective inhibitor of IMP dehydrogenase and a potent antitumor agent, was used. This drug is a C-nucleoside and is taken up into cells presumably through the nucleoside transport system(s) (45, 58). IMP dehydrogenase is the rate-limiting enzyme of de novo GTP biosynthesis (59), and treatment of cells with tiazofurin results in a large decrease in intracellular GTP concentrations (60). In this study, depletion of intracellular GTP pools by treatment with tiazofurin resulted in a significant increase in in situ TG activity. GTP levels in most cells are approximately 100 µM (52), and the Ki for inhibition of tTG by GTP has been reported to be 90-150 µM (4). Treatment of SH-SY5Y cells with 50 µM tiazofurin decreased GTP levels by 75-80%. The resulting GTP levels in response to tiazofurin treatment are thus well below the reported Ki values and therefore are likely to be responsible for the observed tiazofurin-induced increases in in situ TG activity. No loss of cell viability was observed with the doses of tiazofurin that resulted in increased TG activity. This is the first demonstration that GTP may regulate the transamidating activity of TG in situ and suggests that GTP binding influences the in situ interconversion between the transamidating and G protein functions of tTG. Although depletion of intracellular GTP levels resulted in an increase in TG activity, this increase in activity was less than that obtained by increasing intracellular calcium levels. It can be suggested that the more robust response of TG activity in response to calcium-mobilizing agents compared with that seen with GTP depletion is probably due to differences in modulating mechanisms, with GTP regulating the proportion of the protein in the transamidating (versus G-protein) form and calcium directly stimulating the transamidating activity of tTG.

Besides directly modulating TG activity, calcium and GTP also interact in regulating the proteolysis of tTG. Treatment of SH-SY5Y cells with either MTX or tiazofurin resulted in increases in TG activity through different mechanisms. Thus, it was expected that together MTX and tiazofurin would elicit an additive increase in TG activity. In contrast, when tiazofurin-treated cells were challenged with MTX, TG activity was significantly lower than that observed in cells treated with MTX alone. An earlier study that showed that in vitro GTP protected tTG from degradation by trypsin (4) raised the possibility that GTP depletion may increase the degradation of tTG by intracellular proteases. Because in SH-SY5Y cells MTX induces activation of the calcium-activated protease calpain (43), we hypothesized that depletion of GTP resulted in an increase in the degradation of tTG by calpain when intracellular calcium levels were increased by MTX. To test this hypothesis, the membrane-permeable, selective calpain inhibitor Cbz-LLY-DMK (61) was used. Cbz-LLY-DMK blocked the decrease in MTX-stimulated TG activity in tiazofurin-treated cells and inhibited the degradation of tTG. Calpeptin, another calpain-selective inhibitor, gave results almost identical to those obtained with Cbz-LLY-DMK. These results, in combination with the finding that tTG is degraded by purified calpain in vitro (data not shown), indicate that tTG is an endogenous substrate of calpain and that GTP inhibits the degradation of tTG in situ.

Recent studies have demonstrated that differential activation of calcium-mediated processes depends on the rate of calcium entry into the cytosol as well as the spatial and temporal aspects of the calcium increases (e.g. Refs. 62 and 63). Therefore, the ability of several different calcium-mobilizing agents to activate TG in situ was examined. Most intriguingly, stimulation of muscarinic cholinergic receptors with carbachol resulted in an increase in TG activity, and thapsigargin enhanced this response. To our knowledge, this is the first demonstration of a receptor-mediated stimulation of the transamidating activity of tTG. Although circumstantial, activation of TG occurred in the perinuclear regions of the cell (Fig. 6, E and F), containing an abundance of ER membranes where calcium-releasing inositol 1,4,5-trisphosphate receptors are concentrated. Additionally, the MTX-elicited increase in intracellular calcium by activation of ligand and voltage-gated calcium channels (43) stimulates phospholipase activity and inositol phosphate production (44). Thus, it can be speculated that the transamidating activity of tTG is selectively increased by the release of intracellular calcium stores; however, further studies are required.

    ACKNOWLEDGEMENTS

We thank Dr. R. Jope for helpful discussions, Dr. C. Garner for use of the Nikon microscope and photometrics CCD camera, S. Reuver for assistance with image analysis, P. Davis for assistance with GTP measurements, and T. Cox for assistance with manuscript preparation.

    FOOTNOTES

* This study was supported by National Institutes of Health Grant AG12396.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: 1720 7th Ave. S., SC1061, Dept. of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294-0017. Tel.: 205-934-2465; Fax: 205-934-3709; E-mail: gvwj{at}uab.edu.

1 The abbreviations used are: tTG, tissue transglutaminase; TG, transglutaminase; BSA, bovine serum albumin; Cbz-LLY-DMK, N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-tyrosine diazomethyl ketone; ER, endoplasmic reticulum; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; MTX, maitotoxin; RA, retinoic acid; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; BBS, borate-buffered saline; HPLC, high pressure liquid chromatography.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Greenberg, C. S., Birckbichler, P. J., Rice, R. H. (1991) FASEB J. 5, 3071-3077[Abstract/Free Full Text]
  2. Nakaoka, H., Perez, D. M., Baek, K. J., Das, T., Husain, A., Misono, K., Im, M., Graham, R. M. (1994) Science 264, 1593-1596[Medline] [Order article via Infotrieve]
  3. Lee, K. N., Birckbichler, P. J., and Patterson, M. K. (1989) Biochem. Biophys. Res. Commun. 162, 1370-1375[Medline] [Order article via Infotrieve]
  4. Achyuthan, K. E., and Greenberg, C. S. (1987) J. Biol. Chem. 262, 1901-1906[Abstract/Free Full Text]
  5. Feng, J.-F., Rhee, S. G., and Im, M.-J. (1996) J. Biol. Chem. 271, 16451-16454[Abstract/Free Full Text]
  6. Aeschlimann, D., Wetterwald, A., Fleisch, H., and Paulsson, M. (1993) J. Cell Biol. 120, 1461-1470[Abstract]
  7. Eitan, S., and Schwartz, M. (1993) Science 261, 106-108[Medline] [Order article via Infotrieve]
  8. Eitan, S., Solomon, A., Lavie, V., Yoles, E., Hirschberg, D. L., Belkin, M., Schwartz, M. (1994) Science 264, 1764-1768[Medline] [Order article via Infotrieve]
  9. Gentile, V., Thomazy, V., Piacentini, M., Fesus, L., and Davies, P. J. A. (1992) J. Cell Biol. 119, 463-474[Abstract]
  10. Borge, L., Demignot, S., and Adolphe, M. (1996) Biochim. Biophys. Acta 1312, 117-124[Medline] [Order article via Infotrieve]
  11. Hand, D., Campoy, F. J., Clark, S., Fisher, A., and Haynes, L. W. (1993) J. Neurochem. 61, 1064-1072[Medline] [Order article via Infotrieve]
  12. Amendola, A., Gougeon, M.-L., Poccia, F., Bondurand, A., Fesus, L., and Piacentini, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11057-11062[Abstract/Free Full Text]
  13. Johnson, T. S., Knight, C. R., el-Alaoui, S., Mian, S., Rees, R. C., Gentile, V., Davies, P. J., Griffin, M. (1994) Oncogene 9, 2935-2942[Medline] [Order article via Infotrieve]
  14. Hettasch, J. M., Bandarenko, N., Burchette, J. L., Lai, T. S., Marks, J. R., Haroon, Z. A., Peters, K., Dewhirst, M. W., Iglehart, J. D., Greenberg, C. S. (1996) Lab. Invest. 75, 637-645[Medline] [Order article via Infotrieve]
  15. Kojima, S., Inui, T., Muramatsu, H., Suzuki, Y., Kadomatsu, K., Yoshizawa, M., Hirose, S., Kimura, T., Sakakibara, S., and Muramatsu, T. (1997) J. Biol. Chem. 272, 9410-9416[Abstract/Free Full Text]
  16. Nunes, I., Gleizes, P.-E., Metz, C. N., Rifkin, D. B. (1997) J. Cell Biol. 136, 1151-1163[Abstract/Free Full Text]
  17. Raghunath, M., Hopfner, B., Aeschlimann, D., Luthi, U., Meuli, M., Altermatt, S., Gobet, R., Bruckner-Tuderman, L., and Steinmann, B. (1996) J. Clin. Invest. 98, 1174-1184[Abstract/Free Full Text]
  18. Piacentini, M., Martinet, N., Beninati, S., and Folk, J. E. (1988) J. Biol. Chem. 263, 3790-3794[Abstract/Free Full Text]
  19. Beninati, S., and Folk, J. E. (1988) Adv. Exp. Med. Biol. 250, 411-422[Medline] [Order article via Infotrieve]
  20. Hohenadl, C., Mann, K., Mayer, U., Timpl, R., Paulsson, M., and Aeschlimann, D. (1995) J. Biol. Chem. 270, 23415-23420[Abstract/Free Full Text]
  21. Miller, M. L., and Johnson, G. V. W. (1995) J. Neurochem. 65, 1760-1770[Medline] [Order article via Infotrieve]
  22. Ballestar, E., Abad, C., and Francos, L. (1996) J. Biol. Chem. 271, 18817-18824[Abstract/Free Full Text]
  23. Cordella-Miele, E., Miele, L., Beninati, S., and Mukherjee, A. B. (1993) J. Biochem. (Tokyo) 113, 164-173[Abstract]
  24. Baek, K. J., Das, T., Gray, C. D., Desai, S., Hwang, K.-C., Gacchui, R., Ludwig, M., Im, M.-J. (1996) Biochem. J. 35, 2651-2657[CrossRef]
  25. Hwang, K.-C., Gray, C. D., Sweet, W. E., Moravec, C. S., Im, M.-J. (1996) Circulation 94, 718-726[Abstract/Free Full Text]
  26. Suto, N., Ikura, K., and Sasaki, R. (1993) J. Biol. Chem. 268, 7469-7473[Abstract/Free Full Text]
  27. Perry, M. J. M., Mahoney, S.-A., and Haynes, L. W. (1995) Neuroscience 65, 1063-1076[CrossRef][Medline] [Order article via Infotrieve]
  28. Davies, P. J., Murtaugh, M. P., Moore, W. T., Jr., Johnson, G. S., Lucas, D. (1985) J. Biol. Chem. 260, 5166-5174[Abstract]
  29. Piacentini, M., Annicchiarico-Petruzzelli, M., Oliverio, S., Piredda, L., Biedler, J. L., Melino, G. (1992) J. Cancer 52, 271-27820
  30. Benedetti, L., Grignani, F., Scicchitano, B. M., Jetten, A. M., Diverio, D., Lo Coco, F., Avvisati, G., Gambacorti-Passerini, C., Adamo, S., Levin, A. A., Pelicci, P. G., Nervi, C. (1996) Blood 87, 1939-1950[Abstract/Free Full Text]
  31. Kosa, K., Jones, C. S., and De Luca, L. M. (1995) Cancer Res. 55, 4850-4854[Abstract]
  32. Nagy, L., Saydak, M., Shipley, N., Lu, S., Basilion, J. P., Yan, Z. H., Syka, P., Chandraratna, R. A. S., Stein, J. P., Heyman, R. A., Davies, P. J. A. (1996) J. Biol. Chem. 271, 4355-4365[Abstract/Free Full Text]
  33. Chambon, P. (1994) Semin. Cell Biol. 5, 115-125[CrossRef][Medline] [Order article via Infotrieve]
  34. Folk, J. E. (1972) Ann. N. Y. Acad. Sci. 202, 59-76[Medline] [Order article via Infotrieve]
  35. Folk, J. E., and Finlayson, J. S. (1977) Adv. Protein Chem. 31, 1-133[Medline] [Order article via Infotrieve]
  36. Johnson, G. V. W., Cox, T. M., Lockhart, J. P., Zinnerman, M. D., Miller, M. L., Powers, R. E. (1997) Brain Res. 751, 323-329[CrossRef][Medline] [Order article via Infotrieve]
  37. Payne, S. M., and Ames, B. N. (1982) Anal. Biochem. 123, 151-161[Medline] [Order article via Infotrieve]
  38. Mattson, M. P., Cheng, B., Davis, D., Bryant, K., Lieberburg, I., and Rydel, R. E. (1992) J. Neurosci. 12, 376-389[Abstract]
  39. Guttmann, R. P., Elce, J. S., Bell, P. D., Isbell, J. C., Johnson, G. V. W. (1997) J. Biol. Chem. 272, 2005-2012[Abstract/Free Full Text]
  40. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450[Abstract]
  41. Xie, H. Q., and Johnson, G. V. W. (1997) J. Neurochem. 69, 1020-1030[Medline] [Order article via Infotrieve]
  42. Decker, T., and Lohmann-Matthes, M. L. (1988) J. Immunol. Methods 115, 61-69[CrossRef][Medline] [Order article via Infotrieve]
  43. Wang, K. W., Nath, R., Raser, K. J., Hajimohammadreza, I. (1996) Arch. Biochem. Biophys. 331, 208-214[CrossRef][Medline] [Order article via Infotrieve]
  44. Meucci, O., Grimaldi, M., Scorziello, A., Govoni, S., Bergamaschi, S., Yasumoto, T., and Schettini, G. (1992) J. Neurochem. 59, 679-688[Medline] [Order article via Infotrieve]
  45. Weber, G., Prajda, N., Abonyi, M., Look, K. Y., Tricot, G. (1996) Anticancer Res. 16, 3313-3322[Medline] [Order article via Infotrieve]
  46. Singh, U. S., Erickson, J. W., and Cerione, R. A. (1995) Biochemistry 34, 15863-15874[Medline] [Order article via Infotrieve]
  47. Crawford, C., Mason, R. W., Wikstrom, P., and Shaw, E. (1988) Biochem. J. 253, 751-758[Medline] [Order article via Infotrieve]
  48. Morton, A. J., Hammond, C., Mason, W. T., Henderson, G. (1992) Mol. Brain Res. 13, 53-61 [Medline] [Order article via Infotrieve]
  49. Li, X., Song, L., and Jope, R. S. (1996) J. Neurosci. 16, 5914-5922[Abstract/Free Full Text]
  50. Melino, G., Annicchiarico-Petruzzelli, M., Piredda, L., Candi, E., Gentile, V., Davies, P. J. A., Piacentini, M. (1994) Mol. Cell. Biol. 14, 6584-6596[Abstract]
  51. Mian, S., el-Alaoui, S., Lawry, J., Gentile, V., Davies, P. J. A., Griffin, M. (1995) FEBS Lett. 370, 27-31[CrossRef][Medline] [Order article via Infotrieve]
  52. Smethurst, P. A., and Griffin, M. (1996) Biochem. J. 313, 803-808[Medline] [Order article via Infotrieve]
  53. Seiler, N., Delcros, J. G., and Moulinoux, J. P. (1996) Int. J. Biochem. Cell Biol. 28, 843-861[CrossRef][Medline] [Order article via Infotrieve]
  54. Kaouass, M., Audette, M., Ramotar, D., Verma, S., De Montigny, D., Gamache, I., Torossian, K., Poulin, R. (1997) Mol. Cell Biol. 17, 2994-3004[Abstract]
  55. Slaughter, T. F., Achyuthan, K. E., Lai, T. S., Greenberg, C. S. (1992) Anal. Biochem. 205, 166-171[Medline] [Order article via Infotrieve]
  56. Biedler, J. L., Helson, L., and Spengler, B. A. (1973) Cancer Res. 33, 2643-2652[Medline] [Order article via Infotrieve]
  57. Ross, R. A., Spengler, B. A., and Biedler, J. L. (1983) J. Natl. Cancer Inst. 71, 741-747[Medline] [Order article via Infotrieve]
  58. Mitrovic, D. M., Redzic, Z. B., Markovic, I. D., Jovanovic, S. S., Rosic, M. A., Rakic, L. M. (1995) J. Chemother. 7, 543-548[Medline] [Order article via Infotrieve]
  59. Weber, G. N., Nakamura, H., Natsumeda, Y., Szekeres, T., and Nagai, M. (1992) Adv. Enzyme Regul. 32, 57-69[Medline] [Order article via Infotrieve]
  60. Finch, R. A., Revankar, G. R., and Chan, P. K. (1993) J. Biol. Chem. 268, 5823-5827[Abstract/Free Full Text]
  61. Mellgren, R. L., Shaw, E., and Mericle, M. T. (1994) Exp. Cell Res. 215, 164-171[CrossRef][Medline] [Order article via Infotrieve]
  62. Ghosh, A., and Greenberg, M. E. (1995) Science 268, 239-247[Medline] [Order article via Infotrieve]
  63. Golovina, V., and Blaustein, M. P. (1997) Science 275, 1643-1648[Abstract/Free Full Text]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.