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Effect of Inhibiting Vacuolar Acidification on Insulin Signaling in Hepatocytes*

Previous studies have shown that the endosomal apparatus plays an important role in insulin signaling. Inhibition of endosomal acidification leads to a decrease in insulin-insulin receptor kinase (IRK) dissociation and insulin degradation. Thus, vacuolar pH could function as a modulator of insulin signaling in endosomes. In the present study we show that in primary hepatocytes pretreated with bafilomycin, there is an inhibition of vacuolar acidification. Incubation of these cells with insulin was followed by an augmentation of IRK activity but an inhibition of phosphatidylinositol 3-kinase/Akt activity and a decrease in insulin-induced DNA and glycogen synthesis. Bafilomycin treatment inhibited IRK recycling to the plasma membrane without affecting IRK internalization. Impaired IRK recycling correlated with a decrease in insulin signaling. We suggest that inhibiting vacuolar acidification sequesters activated IRKs in an intracellular compartment(s) where signaling is inhibited. This implies that endosomal receptor trafficking plays a role in regulating signal transduction.

Previous studies have shown that the endosomal apparatus plays an important role in insulin signaling. Inhibition of endosomal acidification leads to a decrease in insulin-insulin receptor kinase (IRK) dissociation and insulin degradation. Thus, vacuolar pH could function as a modulator of insulin signaling in endosomes. In the present study we show that in primary hepatocytes pretreated with bafilomycin, there is an inhibition of vacuolar acidification. Incubation of these cells with insulin was followed by an augmentation of IRK activity but an inhibition of phosphatidylinositol 3-kinase/Akt activity and a decrease in insulin-induced DNA and glycogen synthesis. Bafilomycin treatment inhibited IRK recycling to the plasma membrane without affecting IRK internalization. Impaired IRK recycling correlated with a decrease in insulin signaling. We suggest that inhibiting vacuolar acidification sequesters activated IRKs in an intracellular compartment(s) where signaling is inhibited. This implies that endosomal receptor trafficking plays a role in regulating signal transduction.
Insulin binding to its cell surface receptor (IRK) 1 is followed by autophosphorylation, activation, and internalization of the IRK into endosomes (1). Although a proportion of IRK is targeted to lysosomes for degradation, most internalized IRK recycles to the plasma membrane (PM) (2,3). Various studies have shown that signaling by growth factors and hormones, including insulin, occurs at both the cell surface and in endosomes (4 -6). Indeed the exclusive activation of endosomal IRK (5) and epidermal growth factor receptor (7) has been shown to yield substrate tyrosine phosphorylation and signal transduction. It follows that the extent and duration of cell signaling is subject to modulation by endosomal processes (3,8). It has been demonstrated that the endosomal IRK is dephosphorylated by an associated phosphotyrosine phosphatase (3,9). Furthermore, a reduced intraendosomal pH (pH Յ 6) promotes dissociation of internalized insulin-IRK complexes and degradation of insulin (10,11) by an acidic endosomal insulinase recently identified as cathepsin D (12). The acidic pH of endosomes also effects a conformation-dependent inactivation of the IRK (13). Thus, the acidic pH of the endocytic compartment and the IRK-associated phosphotyrosine phosphatase(s) therein play fundamental roles in attenuating insulin signaling.
Weak bases, proton ionophores, and inhibitors of vacuolar ATPases have been used to study the role of acidification in physiological processes (14,15). In liver, chloroquine (a weak base) accumulates in hepatic endosomes leading to the inhibition of both the dissociation of insulin-IRK complexes and insulin degradation (16 -18). This suggests that inhibitors of vacuolar acidification would potentiate insulin signaling from the endosomal compartment, and there are studies consistent with this view. Thus, in rats, chloroquine treatment augmented and prolonged IRK activity in hepatic endosomes (19). Further, the administration of chloroquine to patients with type 2 diabetes mellitus decreased fasting plasma glucose levels, improved glucose tolerance, and increased plasma insulin levels (20 -22). However, it is not clear whether the effects of chloroquine in these patients are associated exclusively with improved insulin sensitivity as opposed to an increase in plasma insulin levels. In contrast, a previous study in adipocytes showed that chloroquine inhibited insulin-induced GLUT4 translocation to the PM (23). In view of the importance of this issue to an understanding of insulin signaling and potentially insulin resistance and in view of the above-noted discrepancies, we examined the role of vacuolar acidification on insulin signaling in primary rat hepatocytes. ). An antibody against tyrosinephosphorylated proteins (PY99) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-p85, anti-IRS1, anti-IRS2, and anti-phospho-GSK-3 (Ser 9/21 ) antibodies were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). An antibody raised against a peptide corresponding to residues 942-969 of the IRK ␤-subunit (␣960) was used for Western blotting and prepared as previously described (25). For immunoprecipitation of IRK, an antibody directed against the ␣-subunit was obtained from the serum of a patient with acanthosis nigricans (1). Protein A-Sepharose was obtained from Amersham Biosciences. Immobilon-P transfer membranes were obtained from Millipore Corp. Canada Ltd. (Mississauga, Canada). Bafilomycin A1, Chloroquine, poly(Glu 4 :Tyr 1 ), and most other chemicals were purchased from Sigma.
Glycogen Synthesis-Glycogen synthesis was determined by incorporation of [U-14 C]glucose into glycogen as previously described (26). Briefly, the hepatocytes (1ϫ10 6 cells) were serum-deprived for 4 h and incubated for 2 h in serum-free medium containing 15 mM [U-14 C]glucose in the presence or absence of bafilomycin A1 (100 nM) and insulin (100 nM). Incubations were stopped by three rapid washes with ice-cold PBS, and the cells were solubilized in 1 ml of 0.1 M NaOH. The samples were then boiled in the presence of 2 mg of carrier glycogen and precipitated overnight in 70% ethanol at Ϫ20°C. After centrifugation, the precipitated glycogen was resuspended in 500 l of water and incubated for 5 min at 70°C. Incorporated radioactivity was determined by scintillation counting as previously described (27) Thymidine Incorporation Assay-Subconfluent hepatocytes were plated on 9.6-cm 2 six-well plates in serum-containing medium for 24 h and then in serum-and growth factor-free medium for an additional 48 h. Thymidine (5 Ci/ml), insulin, and bafilomycin A1 were added to cells as described in the figure legends. After an 18-h incubation, the cells were rinsed twice with 3 ml of cold PBS, incubated for 15 min at 4°C in 10% trichloroacetic acid, solubilized at room temperature in 1 ml of 1 N NaOH, and then transferred to scintillation vials for counting of 3 H in an LKB liquid scintillation counter as described before (24).
PI 3-Kinase Activity-Phosphotyrosine proteins were immunoprecipitated from cell lysates with an ␣PY antibody. The immunoprecipitates were extensively washed, and the protein A-Sepharose pellet was resuspended in 50 l of kinase assay buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5 mM EGTA) containing 0.5 mg/ml phosphatidylinositol and assayed for PI 3-kinase activity as previously described (28).
Determination of the Level of IRK Associated with the PM-Detection of PM-IRK was performed as described previously (29). Briefly, hepatocytes were incubated in the presence or absence of bafilomycin and insulin as noted in figure legends. Thereafter, hepatocytes were washed three times with ice-cold PBS-Ca 2ϩ -Mg 2ϩ (PBS with 0.1 mM CaCl 2 and 1 mM MgCl 2 , pH 7.4), and cell surface proteins were biotinylated by incubation with 0.5 mg/ml of Sulfo-NHS-LC-Biotin (Pierce) in PBS-Ca 2ϩ -Mg 2ϩ for 30 min at 4°C. The reaction was stopped by washing the dishes three times with PBS-Ca 2ϩ -Mg 2ϩ containing 15 mM of glycine. After biotinylation, the cell lysates were prepared as described above, and IRK was immunoprecipitated with an antibody directed against the ␣-subunit of the IRK. The immunoprecipitates were boiled in the presence of Laemmli buffer and subjected to SDS-PAGE. The proteins were FIG. 1. Inhibition of vacuolar acidification by bafilomycin. Primary hepatocytes were incubated at 37°C for 30 min with or without bafilomycin (100 nM) followed by the addition of insulin (100 nM) or buffer alone for an additional 15 min. Following the addition of DAMP, the acidic compartments were visualized by immunofluorescence as described under "Experimental Procedures." FIG. 2. Inhibition by bafilomycin of IRK recycling but not internalization. a, IRK internalization: Primary hepatocytes were incubated with or without bafilomycin (Bafilo, 100 nM) for 30 min at 37°C followed by the addition of insulin (100 nM) for the indicated times. The cell surface proteins were biotinylated as described under "Experimental Procedures." The cells were then lysed, and IRK was immunoprecipitated from these lysates using an antibody to the ␣-subunit of IRK. The immunoprecipitates were boiled in Laemmli sample buffer and subjected to SDS-PAGE. The gel proteins were transferred to Immobilon-P membranes that were probed with an anti-IRK antibody (␣960) or with streptavidin-horseradish peroxidase (Strept) for detection of the total amount of IRK or PM-associated IRK, respectively. To assess specificity of immunoprecipitation, control samples were immunoprecipitated (IP) with normal IgG. The depicted experiment is one of three similar ones. b, IRK recycling. Hepatocytes were incubated with or without bafilomycin (100 nM) as above. Insulin (100 nM) or buffer was added for an additional 10 min to allow internalization of the IRK. The cells were subsequently washed three times with serum-free medium and incubated for an additional 10 min in the absence of insulin to permit recycling of IRK to the PM. The cell surface proteins were biotinylated, and total IRK and PM-associated IRK were determined as indicated above. WB, Western blotting. transferred to Immobilon-P membranes and immunoblotted with ␣960 or streptavidin-horseradish peroxidase (Amersham Biosciences). Streptavidin binds to biotinylated proteins, allowing only the detection of IRK associated with the PM.
Immunofluorescence-Acidic compartments in primary hepatocytes were visualized by immunofluorescence with the DAMP method as indicated by the manufacturer (Oxford Biomedical Research, Oxford, MI). Briefly, the primary hepatocytes were incubated with DAMP (30 M) for 30 min, fixed in 4% paraformaldehyde, washed, and permeabilized by incubation with 0.5% Triton X-100. The fixed hepatocytes were incubated with a monoclonal anti-DNP antibody for 60 min and subsequently washed to remove unbound IgG. The cells were then incubated for 60 min with anti-mouse IgG conjugated to rhodamine and viewed under a Zeiss Axiovert 135 confocal microscope. Photographs were taken using a Zeiss LSM microsystem.

RESULTS
Bafilomycin is a macrolide antibiotic that, at concentrations up to 1 M, specifically inhibits vacuolar ATPases without the side effects induced by proton ionophores and weak bases (15,30). In this study we use bafilomycin to evaluate the effect of inhibiting vacuolar acidification on insulin signaling.
Effect of Bafilomycin on Vacuolar Acidification and Trafficking of IRK-First, we confirmed that bafilomycin inhibits vacuolar acidification by incubating primary hepatocytes with DAMP, a compound that accumulates in acidic vesicles. On performing this incubation, DAMP, detected by immunofluorescence (see "Experimental Procedures"), was found in granular structures in the presence or absence of insulin (Fig. 1, top panels). Preincubation with bafilomycin (100 nM for 30 min) abolished the granular fluorescence almost completely (Fig. 1,  bottom panels), indicating that bafilomycin inhibited vacuolar acidification in primary hepatocytes.
Previous work has shown that inhibition of vacuolar acidifi-

FIG. 4. Bafilomycin increases insulin-induced IRK activity.
Hepatocytes were treated with (E) or without (q) bafilomycin (100 nM) for 30 min at 37°C. Insulin (100 nM) or buffer was then added for an additional 2 or 15 min. IRK was subsequently partially purified from lysates using wheat germ agglutinin columns as described under "Experimental Procedures." a, aliquots of wheat germ agglutinin-purified IRK were subjected to SDS-PAGE, and the proteins were transferred to Immobilon-P membranes. The level of IRK tyrosine phosphorylation and IRK content were determined by immunoblotting using specific antibodies against phosphotyrosine proteins (␣PY) and IRK (␣960), respectively. To quantitate the degree of tyrosine phosphorylation of the IRK, the ␤-subunit bands of IRK and their phosphotyrosine content were determined by scanning densitometry and expressed as a PY/IRK ratio. cation interferes with receptor recycling (31,32). We used cell surface biotinylation to assess the effect of bafilomycin on insulin-induced IRK internalization and recycling to the cell surface. Hepatocytes were preincubated with insulin (100 nM) in the presence or absence of bafilomycin, and cell surface proteins were subsequently labeled with Sulfo-Biotin as described under "Experimental Procedures." The cells were lysed, the IRKs were immunoprecipitated from cell lysates, and both total and PM-associated IRKs were detected by immunoblotting with ␣960 and streptavidin, respectively. It can be seen that bafilomycin did not inhibit insulin-induced IRK internalization (Fig. 2a). Consistent with this was our observation that, following 15 min of incubation with insulin, the increase in the level of IRK in a crude endosomal preparation from hepatocytes was not significantly affected by bafilomycin (analysis by Student's t test, p Ͼ 0.05; data not shown).
To investigate the effect of bafilomycin on IRK recycling, internalization of IRK was promoted by incubating hepatocytes with insulin (100 nM) for 10 min followed by washing to remove ambient insulin and further incubation for 10 min to allow recycling of internalized IRK. In control cells most internalized IRK recycled to the PM in the absence of ambient insulin, whereas in cells incubated with bafilomycin recycling of IRK to the PM was strikingly inhibited (Fig. 2b).
Effect of Bafilomycin on Insulin Action in Primary Hepatocytes-To determine the effect of bafilomycin on insulin action, we measured the effect of insulin on thymidine incorporation and glycogen synthesis in hepatocytes incubated with or with-out bafilomycin. As is evident in Fig. 3, bafilomycin significantly inhibited the effect of insulin on both thymidine incorporation and glycogen synthesis by 40 and 60%, respectively.
Bafilomycin and IRK Activation-We next sought to determine whether the inhibitory effect of bafilomycin on thymidine incorporation and glycogen synthesis corresponded to an impairment in insulin signaling. Pretreatment of hepatocytes with bafilomycin did not modify insulin-induced augmentation of IRK phosphotyrosine content at 2 or 15 min after insulin (Fig. 4a). However, preincubation with bafilomycin did augment IRK activity significantly at 15 but not 2 min following insulin (Fig. 4b). We conclude that bafilomycin pretreatment results in an increase in IRK activity without a proportional increase in IRK phosphotyrosine content (Fig. 4c). In previous work we showed that endosomal IRK is inactivated consequent to a conformational change induced by acidic pH (13). Thus, neutralizing vacuolar pH with bafilomycin could suppress this acidification-dependent conformational change leading to increased IRK activity. This might explain the lack of correlation between IRK activity and tyrosine phosphorylation following bafilomycin treatment.
Bafilomycin and Downstream Signaling Events-We investigated the effect of bafilomycin on insulin-induced downstream signaling events. Hepatocytes were pretreated with or without bafilomycin, and the tyrosine phosphorylation of both IRS1 and IRS2 was assessed at 2 and 15 min after insulin. Bafilomycin had no effect on insulin-augmented IRS1 tyrosine phosphorylation (Fig. 5a, left panel) but significantly inhibited FIG. 5. Bafilomycin inhibits insulin-induced tyrosine phosphorylation of IRS2 but not its association with p85. Primary hepatocytes were treated with bafilomycin (100 nM) for 30 min at 37°C followed by insulin (100 nM) or buffer for 2 or 15 min. IRS1 and IRS2 were immunoprecipitated (IP) from lysates (1 mg of protein) with specific antibodies bound to protein-A. The immunoprecipitates were washed, boiled in Laemmli buffer, and subjected to SDS-PAGE, and the proteins were transferred to Immobilon-P membranes as described under "Experimental Procedures." The level of phosphotyrosine (a) and p85 (b) associated with IRS1 and IRS2 were determined by immunoblotting with ␣PY and ␣p85, respectively. A representative immunoblot at the top of each bar graph is shown. The results are the means Ϯ S.E. of three to five separate experiments. *, p Ͻ 0.005 (versus 15 min of insulin). PY, phosphotyrosine; WB, Western blot.
FIG. 6. Bafilomycin inhibits insulin-induced PI 3-kinase activity. a, hepatocytes were treated with or without bafilomycin (100 nM) for 30 min at 37°C, after which insulin (100 nM) was added for 2 or 15 min. PI 3-kinase activity was measured in ␣IRS1 and ␣IRS2 immunoprecipitates (IP) with [␥-32 P]ATP as described under "Experimental Procedures." An autoradiograph depicting the phosphorylation of phosphatidylinositol in position 3 from an experiment performed in duplicate is shown. b, hepatocytes were treated as indicated above, and PI 3-kinase activity was measured in ␣PY immunoprecipitates. A representative autoradiograph is shown. Autoradiographs were quantified by scanning densitometry, and the results are expressed as the means Ϯ S.E. of three separate experiments, each performed in triplicate. *, p Ͻ 0.01 (versus 15 min of insulin). c, the p85 subunit of PI 3-kinase was immunoprecipitated from hepatocyte lysates (1 mg of protein) with an ␣p85 antibody. The immunoprecipitates were washed, boiled in Laemmli buffer, and subjected to SDS-PAGE, and the proteins were transferred to Immobilon-P membranes as described under "Experimental Procedures." Immunoblotting was performed with ␣PY. A representative immunoblot (one of four separate experiments) is shown. To assess the specificity of Immunoprecipitation, control samples were immunoprecipitated with normal IgG. WB, Western blot.
In response to insulin, tyrosine phosphorylated IRS1 and IRS2 bind p85 (a regulatory subunit of PI 3-kinase), leading to the activation of PI 3-kinase. We thus assessed the effect of bafilomycin pretreatment on the extent of association of p85 with IRS1 and IRS2 following insulin treatment. As seen in Fig. 5b, incubation of hepatocytes with insulin stimulated the association of p85 with both IRS1 and IRS2 immunoprecipitates. Pretreatment with bafilomycin had no effect on the extent of the insulin-induced association (Fig. 5b). We then determined whether insulin-stimulated activation of PI 3-kinase associated with IRS1 or IRS2 is affected by bafilomycin. As seen in Fig. 6a, bafilomycin had no effect on PI 3-kinase activation assessed in anti-IRS1 and -IRS2 immunoprecipitates at 2 min but reduced this activity by 80% in anti-IRS2 immunoprecipitates at 15 min following incubation with insulin. Next we investigated the impact of reduced IRS2 associated PI 3-kinase activity on the total activity. Bafilomycin had no effect on PI 3-kinase activation assessed in anti-phosphotyrosine immunoprecipitates at 2 min but reduced this activity significantly by 60% at 15 min (p Ͻ 0.01 versus insulin alone) following incubation with insulin. As seen in Fig. 6c, a broad band at 160 -180 kDa represents the main phosphotyrosine protein co-immunoprecipitated with p85 in response to insulin. Previous studies in hepatocytes have shown that this band corresponds almost exclusively to IRS1 and IRS2 (33). Thus, in hepatocytes pretreated with bafilomycin, IRS2 is probably the main tyrosine-phosphorylated protein involved in the decrease of PI 3-kinase activity. To a minor extent, insulin increased the association of a 60-kDa phosphotyrosine protein with p85 (Fig.  6c), but bafilomycin did not cause a significant change in this association (four independent experiments; analysis by Student's t test, p Ͼ 0.05 versus insulin alone). Also, 110 -115-and 200-kDa phosphotyrosine proteins associated with p85 (Fig.  6c), but such an association was not increased in response to insulin. In conclusion, bafilomycin increased IRK tyrosine kinase activity but paradoxically decreased PI 3-kinase activity associated with phosphotyrosine proteins in hepatocytes 15 min after insulin. Such a decrease correlated with diminished tyrosine phosphorylation of IRS2 and decreased PI 3-kinase activity associated with IRS2.
Insulin treatment results in a PI 3-kinase-dependent activation of Akt by phosphorylation at specific Ser residues, in particular Ser 473 (34). We therefore determined the effect of bafilomycin on Akt activation by immunoblotting with antibody specific for Akt phosphorylated at Ser 473 . Pretreatment with bafilomycin, in parallel with its effect upon PI 3-kinase, did not affect Akt-Ser 473 phosphorylation at 2 min but inhibited it by 40% (p Ͻ 0.0001 versus insulin alone) at 15 min after insulin (Fig. 7a). Furthermore, bafilomycin inhibited Akt-Ser 473 phosphorylation by 50 -60% with respect to corresponding controls at 0.5, 2, and 4 h after insulin (Fig. 7b). These latter results correlate with the inhibitory effect of bafilomycin on insulin-stimulated glycogen synthesis and thymidine incorporation, both of which determinations entailed longer incubations with insulin (see "Experimental Procedures"). There was no effect of bafilomycin on the level of hepatocyte Akt as determined by immunoblotting (data not shown).
In response to insulin, Akt activation results in augmented phosphorylation of its substrate, GSK-3, with accompanying inactivation of this enzyme, which contributes to insulin-induced activation of glycogen synthase (35). We employed immunoblotting to assess the extent of phosphorylation at Ser 21 and Ser 9 of GSK-3␣ and GSK-3␤, respectively, as a measure of insulin-induced GSK-3 inactivation. In parallel with our observations on PI 3-kinase and Akt, bafilomycin did not affect GSK-3 serine phosphorylation at 2 min after insulin but inhibited it by 20% (p Ͻ 0.05 versus insulin alone) at 15 after insulin (Fig. 7c).
Bafilomycin neutralizes endosomal acidification by inhibiting vacuolar ATPases responsible for maintaining a proton gradient (15). It was therefore of interest to determine whether chloroquine, an acidophilic compound shown to neutralize vacuolar pH by virtue of being a weak base (36), would similarly affect insulin signaling. The preincubation of hepatocytes with 0.1 mM chloroquine increased IRK activity and decreased Akt-Ser 473 phosphorylation at 15 but not at 2 min after insulin (Fig.  8). Chloroquine at 1.0 mM promoted an even greater increase of IRK activity at 15 min after insulin but also increased IRK tyrosine phosphorylation at this time (Fig. 8, a, lower panel,  and b). At this higher dose of chloroquine we observed an inhibition of Akt-Ser 473 phosphorylation at both 2 and 15 min after insulin (Fig. 8c). These results raise the possibility that high concentrations of chloroquine may affect signaling by a mechanism additional to that of vesicular acidification as previously suggested (23,37). In summary, the similarity between the effects of bafilomycin and chloroquine (0.1 mM) on IRK and Akt activation suggests that bafilomycin affects insulin signaling by disrupting vesicular acidification.
Relation of Akt Activation to IRK Recycling-The mechanism by which bafilomycin inhibits insulin signaling at 15 min, but not at earlier times of exposure, could involve an increase in the kinetics of PI 3-kinase/Akt inactivation after maximal activation or a reduction in surface IRK because of the inhibition of FIG. 8. Chloroquine augments insulin-dependent IRK activation but inhibits Akt phosphorylation. Hepatocytes were treated with or without chloroquine (Chl) (0.1 or 1.0 mM) for 1 h at 37°C after which insulin (100 nM) was added for 2 or 15 min. The lysates were prepared, and equal amounts of protein (50 g) were subjected to SDS-PAGE and transferred to Immobilon membranes. The levels of IRK tyrosine phosphorylation and IRK content were determined by immunoblotting using specific antibodies against phosphotyrosine proteins (␣PY) and IRK (␣960), respectively (a). Insulin receptor tyrosine kinase activity (b) was determined as described in the legend of Fig. 4. The level of Akt-Ser 473 phosphorylation (c) was determined as described in the legend of Fig. 7. Each bar represents the mean of two independent experiments Ϯ half the difference. WB, Western blot. IRK recycling. To differentiate between these two possible mechanisms, we studied in more detail the time course of insulin-stimulated Akt-Ser 473 phosphorylation. As seen in Fig. 9a, incubation with insulin resulted in maximal phosphorylation of Akt between 2 and 5 min, a rapid decrease at 8 min, and a subsequent increase at 15 min. Pretreatment with bafilomycin resulted in an insulin-stimulated time course of Akt-Ser 473 phosphorylation, which was greater than control between 2 and 8 min (difference at 8 min, p Ͻ 0.05) but significantly inhibited thereafter (Fig. 9a). These results suggest that, in the presence of ambient insulin, the recycling of IRK is accompanied by a second wave of PI 3-kinase/Akt activation that, corresponding to the inhibition of IRK recycling (Fig. 2b), is impaired by bafilomycin pretreatment of the hepatocytes.
To confirm this hypothesis, hepatocytes were stimulated for 4 min with insulin to allow maximal activation of Akt, followed by washing and further incubation for 11 min in the absence of insulin. By removing insulin from the medium, the contribution of IRK recycling to PI 3-kinase/Akt activation is blocked, and thus, only the kinetics of Akt inactivation will be assessed. As seen in Fig. 9b, hepatocytes washed after 4 min of incubation with insulin showed a reduction in Akt phosphorylation compared with that in cells exposed to insulin for 15 min (Fig.  9b, compare lysates 3 and 4 with lysates 5 and 6). Thus, the level of Akt phosphorylation at 15 min is not exclusively determined by its maximal activation at 4 min following insulin but reflects ongoing exposure of IRK to ambient insulin. Also bafilomycin did not affect the level of Akt phosphorylation in hepatocytes washed 4 min after exposure to insulin (Fig. 9b, compare lysates 3 and 4 with lysates 9 and 10). These results show that the kinetics of Akt-Ser 473 dephosphorylation is not affected by bafilomycin and confirm that a decrease in IRK recycling is responsible for the inhibition of PI 3-kinase/Akt activity observed at later times of insulin signaling. DISCUSSION Our results show that the inhibition of vacuolar acidification by bafilomycin results in decreased insulin-dependent activation of PI 3-kinase/Akt, thymidine incorporation, and glycogen synthesis. These findings conform to previous studies in hepatocytes showing that insulin-stimulated thymidine incorporation and glycogen synthesis are both dependent on PI 3-kinase activation (24,38).
Paradoxically, we found that the bafilomycin-induced decrease in PI 3-kinase/Akt activity was accompanied by a corresponding augmentation of IRK activity, and both of these effects of bafilomycin were observed only at later times (15 min) after insulin stimulation. We propose that the different effects of bafilomycin on insulin signaling at early and late times reflect differences in the cellular location of IRK at 2 and 15 min after insulin stimulation. The lack of an inhibitory effect of bafilomycin on insulin signaling at 2 min reflects the high proportion of IRK at the PM (Fig. 1a) and in early endosomal elements (25) at this time. In contrast, the effects of bafilomycin on IRK activity and signaling at 15 min after insulin coincide with the intracellular accumulation of receptor (Fig. 2a). Bafilomycin did not interfere with the insulin-stimulated internalization of IRK but inhibited the recycling of IRK to the PM (Fig. 2). This is consistent with the view that IRK is being sequestered into an intracellular subcompartment where signaling is blunted. This study also shows that IRK recycling is critical for PI 3-kinase/Akt activation (Fig. 9b), leading us to conclude that continuous recycling of IRK between internal compartments and PM is necessary for sustained insulin signaling.
The comparable effect of chloroquine to augment insulin-dependent IRK activation while inhibiting Akt activation at later times after insulin stimulation (Fig. 8) argues that these responses are consequences of the inhibition of vacuolar acidification. Interestingly at higher doses other effects of chloroquine were observed including the inhibition of Akt activity at 2 min after insulin. We suggest that this could result from nonacidotropic effects of chloroquine such as its capacity, at higher concentrations, to effect vacuolar swelling (15).
Previous work has shown that bafilomycin inhibited the trafficking from late endosomes to lysosomes of the fluid phase marker, horseradish peroxidase (39). Also, we have previously shown that chloroquine promotes the accumulation of insulin in a late endosomal fraction derived from liver (16). In hepatocytes, late endosomes are filled with tracers between 10 and 15 min (40). Thus, in the presence of bafilomycin, activated IRK could be sorted into the luminal vesicles of late endosomes and thereby segregated away from the cytoplasm, preventing interactions with downstream signaling proteins (41).
This model is consistent with our finding that the increase of IRK activity in response to bafilomycin was accompanied by a decrease in IRS2 tyrosine phosphorylation and IRS2-associated PI 3-kinase activity only after 15 min of incubation with insulin in hepatocytes preincubated with bafilomycin. A previous study has identified IRS2 as the main effector of the metabolic and growth-promoting actions of insulin in hepatocytes (33). Our data, together with this previous work, assign an important role to IRS2 in promoting actions of insulin in hepatocytes probably through the recruitment and activation of PI 3-kinase. However, we found that the decrease in IRS2 tyrosine phosphorylation caused by bafilomycin did not reduce the level FIG. 9. Inhibition of insulin signaling by bafilomycin linked to inhibition of IRK recycling. a, hepatocytes were treated with (q) or without (E) bafilomycin (100 nM), followed by insulin (100 nM) for the indicated times. The level of Akt Ser 473 phosphorylation was determined as described in the legend of Fig. 7. The results are the mean Ϯ S.E. of three to ten independent experiments. *, p Ͻ 0.0001; #, p Ͻ 0.05 (versus insulin alone). b, hepatocytes were incubated as above with or without bafilomycin for 30 min; followed by insulin for 4 min. The cells were then washed with serum-free media and incubated for a further 11 min in the absence of insulin. Also, the hepatocytes were incubated with or without bafilomycin (100 nM for 30 min) followed by insulin (100 nM) for an additional 4 or 15 min. Akt-Ser 473 phosphorylation was visualized as indicated in the legend to Fig. 7. WB, Western blot. of association of p85 with IRS2. A possible explanation for this observation could be that the diminished phosphorylation of IRS2 did not affect the recruitment of p85 but did affect the association of the SH2 domains of p85 with IRS2 leading to decreased PI 3-kinase activity. In agreement with this, it has been shown that full activation of PI 3-kinase by tyrosinephosphorylated proteins required occupancy of both vicinal SH2 domains in p85 (42).
In previous work we showed that inhibition of endosomal acidification with chloroquine inhibited both insulin degradation and dissociation of insulin-IRK complexes within endosomes (18), with the consequent augmentation of IRK activity in this compartment (19). Because a critical role for endosomal IRK in insulin signaling has been clearly demonstrated (5), it was postulated that the inhibition of vacuolar acidification might potentiate insulin signaling from endosomes (19). Our results support this view at early times following insulin administration. Thus, in the present study, bafilomycin significantly increased Akt activity when hepatocytes were incubated with insulin for 8 min (Fig. 8a). This suggests that bafilomycin augmented insulin signaling from early endosomes and that the subsequent accumulation of IRK into a "nonfunctional" compartment resulted in the attenuation of insulin signaling at 15 min.
Our finding is in agreement with previous studies where it has been shown that bafilomycin abolished proliferation of various cell lines (43) as well as mitogen-induced DNA synthesis in 3T3 fibroblasts (44). Of great interest is the study showing that inhibition of an endosomal proteinase, responsible for IGF-1 degradation, increased the tyrosine phosphorylation of IGF-1 receptors in response to IGF-1 but inhibited both the recycling of IGF-1 receptors to PM and IGF-1-induced DNA synthesis (45). These observations together with our findings suggest that the inhibition in endosomes of hormone/growth factor dissociation from its receptor results in the prevention of receptor recycling, sequestration of the complex, and inhibition of signaling.
Several observations from a clinical setting may be relevant to this idea of inactivation of receptor function through sequestration of receptors. Thus, transformed lymphoblasts from subjects with type 2 diabetes mellitus showed impaired intracellular dissociation of insulin-IRK complexes, insulin degradation, and recycling of the IRK when compared with cells from normal subjects, raising the possibility that these defects might underly the insulin resistance seen in these patients (46). Also it has recently been observed that the sulfonylurea glimepiride improved insulin action in hepatoma cells and correspondingly increased the intracellular dissociation of insulin-IRK complexes, the degradation of insulin, and the recycling of internalized IRK in these cells (47). It is tempting to consider that a defect in the vacuolar system responsible for mediating insulin action (endosomes) and insulin processing and secretion (trans Golgi vesicles) may explain both the insulin resistance and impaired ␤ cell function of type 2 diabetes mellitus.
In summary, we have found that the inhibition of endosomal acidification blocks IRK recycling and reduces insulin signaling at later times. We propose that this arises as a result of the sequestration of activated receptors such that they no longer can influence downstream signaling elements. Thus, a continuous trafficking of IRK between endosomes and PM is required to sustain insulin signaling. These observations suggest that disturbances in the trafficking of IRK and possibly other receptors within the endosomal system may have important consequences for the pathogenesis of disease.


extfieldsofstudy: Biology Medicine

text:
A novel transcription regulatory complex containing death domain-associated protein and the ATR-X syndrome protein.

Death domain-associated protein (Daxx) is a multi-functional protein that modulates both apoptosis and transcription. Within the nucleus, Daxx is a component of the promyelocytic leukemia protein (PML) nuclear bodies (NBs) and interacts with a number of transcription factors, yet its precise role in transcription remains elusive. To further define the function of Daxx, we have isolated its interacting proteins in the nucleus using epitope-tagged affinity purification and identified X-linked mental retardation and alpha-thalassaemia syndrome protein (ATRX), a putative member of the SNF2 family of ATP-dependent chromatin remodeling proteins that is mutated in several X-linked mental retardation disorders. We show that substantial amounts of endogenous Daxx and ATRX exist in a nuclear complex. Daxx binds to ATRX through its paired amphipathic alpha helices domains. ATRX has ATPase activity that is stimulated by mononucleosomes, and patient mutations in the ATPase domain attenuate this activity. ATRX strongly represses transcription when tethered to a promoter. Daxx does not affect the ATPase activity of ATRX, however, it alleviates its transcription repression activity. In addition, ATRX is found in the PML-NBs, and this localization is mediated by Daxx. These results show that the ATRX.Daxx complex is a novel ATP-dependent chromatin-remodeling complex, with ATRX being the core ATPase subunit and Daxx being the targeting subunit. Moreover, the localization of ATRX to the PML-NBs supports the notion that these structures may play an important role in transcription regulation.

Daxx 1 was initially identified as a pro-apoptotic molecule that binds to the intracellular tail of the death receptor CD95 (Fas/Apo-1) and enhances apoptosis through the induction of the c-Jun NH 2 -terminal kinase (JNK) pathway (1). Daxx associates with apoptosis signal-regulating kinase 1, an apical kinase of the JNK pathway, causing it to become active (2). Subsequent studies have revealed a role for Daxx in apoptosis induced by other stimuli, including transforming growth factor-␤ and interferons (3,4). Despite these cytoplasmic interactions, Daxx is mainly found in the nucleus, and its cytosolic translocation appears to be regulated. For example, the translocation of Daxx to the cytoplasm can be induced by CD95 engagement in a heat shock protein 27-inhibitable manner (5). In addition, ASK1 can enhance the cytosolic localization of Daxx (6). On the contrary, accumulation of Daxx in the nucleus is observed during the activation of splenocytes (7).
In the nucleus, Daxx is found in the promyelocytic leukemia protein nuclear bodies (PML-NBs) (8 -10), macromolecular complexes that regulate a wide range of cellular processes (11,12). The PML-NBs are dispersed in over 95% of cases of acute promyelocytic leukemia (APL) by the PML-retinoid acid receptor fusion protein generated by a t (15,17) translocation. Treatment of APL patients with all-trans-retinoid acid or arsenic oxide induces degradation of the fusion protein, restoration of normal PML-NB appearance, and concomitant remission of the disease, implicating a critical function of the PML-NBs in protecting against APL pathogenesis (11)(12)(13). In addition, the PML-NBs are disrupted by various viral proteins and increase in both size and number in response to anti-viral cytokine interferons that up-regulate PML-NB components, suggesting a role for the PML-NBs in anti-viral responses (14). Furthermore, cells deficient in PML are resistant to a wide range of apoptotic stimuli, including UV radiation and engagement of CD95 (15), and Daxx appears to be able to regulate apoptosis from the PML-NBs (7,8). How the PML-NBs function in tumor suppression, anti-viral responses, and apoptosis is not well defined. Several cellular proteins have been found in the PML-NBs in addition to PML and Daxx, including p53, pRb, Sp100, SUMO (small ubiquitin-like modifier), CBP, HAUSP, and BLM, and a role for the PML-NBs in transcription has been proposed (16). However, a better understanding of PML-NB function requires the identification of additional key components that associate with these structures.
Daxx has been shown to be a transcription regulatory protein and to associate with several sequence-specific transcription factors, such as Pax3, Pax5, ETS1, and the glucocorticoid re-ceptor (17)(18)(19)(20)(21). The mechanism by which Daxx regulates transcription is not well understood, but it is conceivable that Daxx may associate with chromatin-remodeling complexes. Indeed, the association of Daxx with histone deacetylase chromatin remodeling complexes has been reported (18,22). It remains to be determined whether Daxx also engages the other major class of chromatin remodeling molecules, the ATP-dependent remodeling complexes.
To further define the function of Daxx, we have devised a convenient affinity purification strategy to isolate Daxx-associated proteins. Here, we describe the identification and characterization of ATRX as a Daxx-interacting protein. ATRX is a large protein of ϳ280 kDa that contains a putative ATPase/helicase domain homologous to that found in members of the SNF2 family of chromatin-remodeling proteins. In addition, it also contains a plant homeodomain (PHD) zinc finger domain, which is most similar to the DNA methyltransferase 3 family of proteins. Mutations in the X-linked ATRX gene are most commonly located within the putative ATPase/helicase domain and the PHD motif causing a severe mental retardation syndrome that is associated with urogenital abnormalities, facial dysmorphisms, and frequently ␣-thalassaemia (the ATR-X syndrome) (23)(24)(25). Despite its similarity to chromatin-remodeling proteins, the precise function of ATRX remains largely unknown. We show that endogenous Daxx and ATRX are present in a nuclear complex. Daxx interacts with ATRX through its NH 2 -terminal paired amphipathic alpha helices (PAH) domains. In addition, we demonstrate that ATRX possesses mononucleosome-stimulated ATPase activity, and patient mutations attenuate this activity. Furthermore, we demonstrate that ATRX is localized to the PML-NBs. Daxx recruits ATRX to the PML-NBs and inhibits ATRX-mediated transcription regulation. These results suggest that the ATRX⅐Daxx complex is a novel chromatin remodeling complex and that Daxx may regulate ATRX activity by altering its cellular localization. The localization of ATRX to the PML-NBs also supports the notion that the PML-NBs regulate diverse cellular processes by modulating transcription.
Expression Plasmids-For expression in mammalian cells, fulllength Daxx protein and the deletion mutants of Daxx and ATRX were fused to an NH 2 -terminal FLAG tag in pRK5 (27). pGal, expressing the DNA binding domain of Gal4 (amino acids 1-148), and pGal-ATRX, expressing a fusion of the Gal4 DNA-binding domain of ATRX, were also made in pRK5. The ATRX-GFP fusion plasmids were made in either pEGFP-C2 (for the full-length protein) or pEGFP-C1 (for deletion mutants) (Clontech), whereas RFP-PML was made in pdsRed-N1 (Clontech). For expression in insect cells, human ATRX and ISWI proteins SNF2H and SNF2L were cloned into pBacPAK (Clontech) with either a COOH-terminal HA tag (for ATRX) or an NH 2 -terminal FLAG tag (for SNF2H and SNF2L). The ATRX point mutations were made using the XL site-directed mutagenesis kit (Stratagene) according to the manufacturer's instruction and verified by DNA sequencing. The following mutations were made: PHD, the common R246C mutation located in the PHD zinc finger domain; PED3 and PED23, the Y2084H and D2035V mutations in the putative ATPase/helicase domain which have been previously described (23); Mu, a K1600R mutation in the conserved ATP binding site.
Biochemical Identification of Daxx-binding Proteins-To prepare nuclear extracts, human HeLa S3 cells from 10-liter suspension culture (obtained from the National Cell Culture Center) were lysed in five cell volumes of buffer A (20 mM HEPES-NaOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). Cells were further disrupted by Dounce homogenization, and the nuclei were collected by centrifugation at 1000 ϫ g for 10 min. The nuclei were suspended in buffer B (20 mM HEPES, pH 8.0, 25% glycerol, 1.5 mM MgCl 2 , 400 mM NaCl, 0.2 mM EDTA), and the soluble fraction was diluted 1:1 with a low salt buffer (10 mM HEPES, 1.5 mM MgCl 2 , 10 mM KCl, 0.2 mM EDTA) to yield nuclear extracts. To generate recombinant FLAG-Daxx protein in mammalian cells, FLAG-Daxx/pRK5 or the control vector FLAG/pRK5 was transfected into HeLa cells seeded in thirteen 10-cm plates with 5 g of DNA/plate by the calcium phosphate precipitation method. Twenty-four hours after transfection, cells were washed with ice-cold phosphate-buffered saline and lysed with 1 ml of IP-lysis buffer (27). Cell lysates were incubated with M2-agarose beads for 4 h. After extensive washing, the beads were incubated overnight with 10 mg of HeLa nuclear extracts. The beads were washed four times with IP-lysis buffer and three times with buffer C (10 mM Tris-HCl, pH 7.9, 100 mM KCl, 10% glycerol, 0.2 mM EDTA). The bound proteins were eluted with 100 l of buffer C containing 100 g/ml FLAG peptide for 1 h. The eluted proteins were stored at Ϫ80°C. An aliquot of eluted proteins was resolved on SDS-PAGE gel, and Daxx-associated proteins were analyzed by mass spectrometry.
Gel Filtration Chromatography and Co-immunoprecipitation-Ten milligrams of HeLa S3 nuclear extract were separated on a Superose 6 column driven by an Amersham Biosciences AKTA fast protein liquid chromatography system. Proteins in various fractions were precipitated with trichloroacetic acid and analyzed by SDS-PAGE and immunoblotting. To detect the interaction between endogenous ATRX and Daxx, about 2 ϫ 10 7 HEK293T cells were lysed in IP-lysis buffer. Cell extracts were incubated with the indicated antibodies, followed by incubation with protein A beads. The immunoprecipitated proteins were subjected to SDS-PAGE and immunoblotting analyses. To map the interaction domain on Daxx, FLAG-tagged wild-type Daxx and various mutant Daxx proteins were expressed in 293T cells. The cell extracts were immunoprecipitated with M2 beads and endogenous ATRX protein bound to the beads was detected by immunoblotting. Reciprocally, various FLAG-tagged ATRX constructs were expressed in 293T cells, and their ability to associate with endogenous Daxx was examined by immunoprecipitation assays.
Expression and Purification of Recombinant ATRX, SNF2H, and SNF2L-The pBacPak expression plasmids were co-transfected with BacPAK6 viral DNA into insect Sf21 cells (Clontech) to generate recombinant baculoviruses according to the manufacturer's instruction. Following several rounds of amplification, the recombinant virus was used to infect insect Sf21 cells. Forty-eight hours post-infection, the cells were harvested and recombinant proteins were purified as previously described with modifications (28). The cell pellet was suspended in lysis buffer (20 mM HEPES, pH 7.5, 0.5 M NaCl, 10 mM KCl, 2 mM MgCl 2 , 20% of glycerol, 0.2 mM EDTA, 0.2 mM dithiothreitol, 0.01% Nonidet P-40, 0.2 mM phenylmethylsulfonyl fluoride, and protease inhibitors) with or without DNase I (100 units/ml) and homogenized thoroughly in a Dounce homogenizer (firm pestle). All purification steps were carried out at 4°C. The cell lysate was centrifuged at 15,000 ϫ g for 10 min. The recombinant proteins in the supernatant were affinitypurified using either Fxnp5 plus protein G-Sepharose beads or anti-HA agarose (for ATRX), or with anti-FLAG M2 agarose beads (for SNFH and SNF2L proteins). After extensive washing, the bound proteins were eluted with either 200 g/ml HA or FLAG peptide or with 100 mM glycine (pH 3.2). The protein was frozen in dry ice in small aliquots (20 -50 l) and stored at Ϫ80°C.
ATPase Assay-The ATPase activities of recombinant human ATRX, SNF2H, and SNF2L were measured as previously described with modifications (29). Purified recombinant proteins (5-200 ng) were incubated with 0.25 Ci of [␥-33 P]ATP (3000 Ci/mmol), plus 2.94 nM pUC19 plasmid DNA (100 ng) or 0.83 pmol (250 ng) of mononucleosomes where indicated, in a total volume of 20 l of ATPase assay buffer (25 mM HEPES-KCl, pH 7.5, 5% glycerol, 20 mM KCl, 5 mM MgCl 2 , 25 g/ml bovine serum albumin, 0.5 mM dithiothreitol, 1 M unlabeled ATP, and 0.005% Nonidet P-40). The mixture was stopped after incubation at 27°C for 20 min by adding 2 l of 0.5 M EDTA (pH 8.0). One microliter of the reaction mixture was separated by thin layer chromatography with a poly(ethyleneimine)-cellulose plate (J.T. Baker) in 0.5 M LiCl and 1 M acetic acid. The ratio of free phosphate to ATP was measured by liquid scintillation counting.
Immunofluorescence-HEK293T or HeLa cells were seeded on cover slips placed in 24-well plates. When indicated, cells were treated with interferon-␥ at 5 ng/ml concentration for 24 h. Transfection was performed by the calcium phosphate precipitation method. Cells were fixed with 1% paraformaldehyde for 15 min at room temperature and treated with 0.2% Triton X-100 for 15 min on ice. Cells were then incubated for 1 h with anti-Daxx (M-112), anti-PML (PG-M3), or anti-ATRX (H-300), as indicated, followed by incubation with fluorescein isothiocyanate-or Texas Red-conjugated secondary antibodies. Fluorescence signals were analyzed on a Bio-Rad 1024-ES confocal laser-scanning microscope unless otherwise indicated.
Reporter Gene Assays-HEK293T cells were seeded in 24-well plates and transfected on the second day by the calcium phosphate precipitation method, with 0.4 g of 5xGal-TK-luciferase reporter gene plasmid (30), 50 ng of pCMV-␤-galactosidase, and various amounts of pGal, pGal-ATRX, and Daxx. The total amount of DNA was made constant by adding the pRK5 vector. Twenty-four hours after transfection, cells were harvested and assayed for luciferase activity with a firefly luciferase system (Promega), according to the manufacturer's instruction. Luciferase activities were normalized on the basis of the activities of the co-transfected ␤-galactosidase. Data shown are representative of three independent experiments done in duplicate.

Identification of ATRX as a Novel Daxx-interacting Pro-
tein-To identify Daxx-associated proteins, we expressed a FLAG-tagged Daxx protein (FLAG-Daxx) in HeLa cells and affinity-purified the protein using anti-FLAG monoclonal antibody M2 beads. The immobilized FLAG-Daxx was then incubated with a large quantity of HeLa nuclear extract to identify its interacting proteins. This approach has two major advantages. First, the bait protein is made in mammalian cells and is therefore likely to have the correct post-translational modification. Second, it avoids the lengthy (and sometimes unachievable) process of establishing stable transfectants. As shown in Fig. 1A, a protein around 300 kDa in size in the HeLa nuclear extract specifically associated with the FLAG-Daxx beads but not the control beads. This protein was analyzed by mass spectrometry, and thirteen peptide sequences were obtained, all of which matched the sequence of ATRX (Fig. 1A).
To confirm the Daxx⅐ATRX interaction in another cell line as well as the interaction between the endogenous proteins, we performed immunoprecipitation assays using extracts of hu-man 293T cells. Endogenous Daxx was immunoprecipitated, and ATRX was readily detected by Western blot analysis in the anti-Daxx immunoprecipitates but not in control immunoprecipitates with an unrelated antibody (Fig. 1B, lane 2 versus 1). Reciprocally, Daxx was present in the anti-ATRX immunoprecipitates (lane 3). These assays also revealed that a significant portion of endogenous Daxx and ATRX are in the same complex (data not shown). To further characterize the Daxx⅐ATRX complex, we fractionated HeLa cell nuclear extracts using a gel filtration column and analyzed fractions from the column by Western blot analysis with anti-ATRX and anti-Daxx antibodies. The Daxx and ATRX proteins were co-eluted in a complex of ϳ350 kDa, suggesting a stoichiometric ratio of Daxx and ATRX in the complex. Recently, ATRX was shown to be present in a larger (1 MDa) complex that contained six polypeptides, including Daxx (31). The differences we observed are likely attributable to the sensitivity of the complex to differing salt concentrations used for purification.
Daxx Associates with ATRX via its NH 2 -terminal PAH Domains-To delineate the region of Daxx that mediates its interaction with ATRX, we constructed various deletion mutants of Daxx, each tagged with an NH 2 -terminal FLAG epitope ( Fig.  2A). The FLAG-Daxx proteins were transiently expressed in 293T cells and precipitated with anti-FLAG M2 beads, and the presence of endogenous ATRX in the immunoprecipitates was examined by Western blotting. As shown in Fig. 2B, all Daxx mutants that contained the NH 2 -terminal 160 amino acids, including the first paired amphipathic helix (PAH), were able to associate with ATRX (lanes 2-6), albeit the interaction was stronger if the second PAH domain was also present (amino acids 1-260, lanes 2-5 versus lane 6). In contrast, all of the mutations that were missing the first PAH domain failed to bind to ATRX (lanes 7-9). Together, these results demonstrate that the NH 2 -terminal PAH region of Daxx mediates its interaction with ATRX. Notably, this is the first indication of a function for the PAH domains, as all of the Daxx-interacting proteins previously reported in the literature associate with the COOH region of Daxx (1,3,(17)(18)(19)(20)(21).
Mapping the Daxx-interacting Region of ATRX-To determine the Daxx-interacting domain of ATRX, we generated various FLAG-tagged ATRX deletion mutants (Fig. 3A). Our initial analysis demonstrated that both halves of the ATRX protein can interact with Daxx, indicating that ATRX may contain two distinct Daxx-interacting domains (Fig. 3B, lanes 2  and 7). Further analysis revealed that all of the ATRX fragments containing amino acids 1189 -1326 interacted well with Daxx, whereas the COOH fragments of ATRX that were missing these amino acids failed to do so (lanes 11-14). Thus, amino acids 1189 -1326 constitute a Daxx-interacting domain. In addition, the region between amino acids 321 and 865 may contain a second Daxx-interacting domain (lanes 4 and 9). However, the first domain bound to Daxx more strongly than the second one and therefore may be the domain mainly responsible for the ATRX⅐Daxx interaction (lanes 11, 12, and 14 versus lane 9). In contrast, two previously identified domains of ATRX, the NH 2 -terminal PHD domain and the COOH-terminal ATPase/helicase domains, are not required for the interaction with Daxx (Fig. 3B).

ATRX Possesses ATPase Activity That Is Stimulated by Nucleosomes-The
ATPase/helicase domain of ATRX shares significant homology with those found in members of the SNF2 family of chromatin-remodeling proteins. We therefore sought to determine whether the ATRX protein had ATPase activity. We expressed recombinant ATRX and, for comparison, recombinant mammalian ISWI proteins-SNF2H and SNF2L-in insect cells using baculovirus expression plasmids. The recombinant ATRX protein was purified by an anti-HA affinity column and analyzed by SDS-PAGE followed by silver staining and immunoblotting with either anti-ATRX or anti-HA antibody (Fig. 4A, lanes 3, 6, and 9). Similarly, the recombinant SNF2H and SNF2L proteins containing an NH 2 -terminal FLAG tag were expressed and purified to homogeneity by affinity chromatography (Fig. 4B). As shown in Fig. 4C, ATRX hydrolyzed ATP in a dose-dependent manner (lanes 10 -13 versus 1). To determine the relative specific activities of the different proteins, we plotted the dose-dependent ATPase activities of the purified proteins from three independent preparations and found that the ability of ATRX to hydrolyze [␥-33 P]ATP, based on the molar concentration of protein, was only slightly less than that of SNF2H but significantly higher than the ATPase activity of SNF2L (Fig. 4D). It is clear that the observed ATPase activity originated from the ATRX protein as opposed to one or more contaminant proteins in the preparation, because the activity was either diminished or completely impaired when ATRX mutations were introduced (see below).
Previous studies have shown that the ATPase activity of both the SNF/SWI and the ISWI subfamilies of chromatin remodeling molecules is stimulated by DNA. However, the responses of these molecules to naked and mononucleosomal DNA are different. SNF/SWI proteins are stimulated equally well by both types of DNA, whereas ISWI proteins are stimulated only by nucleosomes (32). To characterize the effects of these DNA substrates on the ATPase activity of ATRX, we examined whether naked DNA or mononucleosomes could stimulate the ATPase activity of ATRX. The ATPase activity of ATRX approximately doubled in response to mononucleosomes but was not stimulated by plasmid DNA (Fig. 5A, lanes 10, 11 versus 9). The effect of the mononucleosomes, albeit moderate, was very reproducible. In addition, the response of ATRX alone was slightly different than the activity of the ATRX⅐Daxx complex, which could be stimulated equally well by both naked DNA and chromatin (31). As a control, we examined the ATPase activity of the two recombinant mammalian ISWI proteins in the presence of either DNA or mononucleosomes and observed that it was consistent with previous studies, with the ATPase activity of SNF2H being stimulated by both and the ATPase activity of SNF2L being only enhanced by mononucleosomes (Fig. 5A,  lanes 1-7) (32).
ATR-X Syndrome-associated Mutations Impair the ATRX ATPase Activity-ATR-X syndrome is associated with a variety of mutations in the ATRX gene, suggesting that the prevailing disease mechanism is the loss of ATRX protein function. However, the absence of mutations in highly conserved residues within the ATPase domain implies that such changes to this domain would be lethal. Thus, patient mutations may only attenuate activity without abolishing it altogether. To distinguish whether known mutations abolish or attenuate ATPase activity, we generated recombinant ATRX proteins harboring known mutations within the ATPase domain (Ped3-Y2084H and Ped23-D2035V), an ATPase-dead mutant (Mu-K1600R), and as a control, the common R246C mutation located within the PHD domain (PHD). All mutant proteins were expressed in insect cells, and three different preparations were purified to homogeneity before examining ATPase activity. Although the point mutation in the PHD domain had no effect on ATPase activity, mutations in the ATPase domain severely impaired activity (Fig. 5B, lanes 3-5). As expected, the Lys-to-Arg mutation in the catalytic site completely abolished the ATPase activity (lane 6). These results demonstrate that patient muta- tions within the ATPase domain severely attenuate activity and confirm that this domain is critical for ATRX function. Moreover, it suggests that mutations in other domains of the protein do not modulate ATPase activity but likely alter ATRX function through alternative means and that abolishing ATPase activity completely is likely non-viable.
The ATPase Activity of ATRX Is Not Affected by Daxx-Having shown that ATRX has ATPase activity that is defective in certain ATR-X patients, we examined whether Daxx regulates the ATPase activity of ATRX. FLAG-tagged Daxx and DaxxC, the latter missing the ATRX interacting domain, were expressed in mammalian cells and affinity-purified to homogeneity (Fig. 5C, left panel). When these Daxx proteins were added to the ATPase assay reaction, no effect on ATP hydrolysis was observed (Fig. 5C, lanes 3 and 4 versus 1). Thus, Daxx does not appear to modulate the ATPase activity of ATRX.
ATRX Represses Transcription When Tethered to DNA and This Transcriptional Repression Is Alleviated by Daxx-We next analyzed the effect of Daxx on ATRX-mediated transcriptional regulation. Because ATRX is a chromatin-remodeling molecule, we speculated that tethering ATRX to a promoter would affect transcription. We fused ATRX to the Gal4 DNAbinding domain (Gal-ATRX) and introduced this fusion into 293T cells together with a luciferase reporter plasmid containing five Gal4 DNA-binding sites upstream of the thymidine kinase (TK) promoter. Gal-ATRX strongly inhibited expression of the TK promoter-driven luciferase (Fig. 6A, columns 3-6  versus 1 and 2). This transcription repression was dependent on the targeting of ATRX to the promoter, because FLAGtagged ATRX failed to inhibit luciferase expression (column 7).
Thus, ATRX can function as a transcription repressor. This is consistent with previous findings that ATP-dependent chromatin-remodeling molecules can repress the expression of some genes while activating the expression of others (33). When Daxx was included in this reporter assay, transcription repression by ATRX was reduced in a Daxx dose-dependent manner (Fig. 6B, columns 2 and 3 versus 1). Therefore, Daxx inhibits the function of ATRX in transcription regulation.
Localization of ATRX to the PML-NBs-Because Daxx had no effect on the ATPase activity of ATRX but still inhibited ATRXmediated transcriptional activity, we investigated the possibility that Daxx alters the cellular localization of ATRX. Given that Daxx is found in the PML-NBs, we tested whether ATRX is also localized to these nuclear bodies and, if so, whether this localization is dependent on Daxx. Endogenous ATRX in HeLa cells showed a diffuse staining pattern with several prominent nuclear speckles that nicely co-stained with an anti-PML antibody (Fig. 7A, panels a-c). In addition, consistent with previous studies, the size of the PML-NBs was enhanced upon interferon treatment, and concomitantly, more ATRX was found to be present in the PML-NBs (Fig. 7A, panels d-f). These results confirm that ATRX is a PML-NB component.
Daxx Targets ATRX to the PML-NBs-To investigate the role of Daxx in the targeting of ATRX to the PML-NBs, we first tested whether Daxx enhances the PML-NB localization of ATRX. Expression of a GFP fusion of ATRX in 293T cells showed a pattern of nuclear dots against a diffuse nuclear distribution that was similar to the endogenous protein. The GFP-ATRX dots were co-localized with Daxx in the PML-NBs (Fig. 7B, panels a-c). Notably, this localization was enhanced when Daxx was co-transfected with GFP-ATRX (panels d-f). To facilitate PML-NB targeting of Daxx, we included PML in the experiments as previous studies have shown that PML recruits Daxx to the nuclear bodies (7,9,18). We next analyzed the structural determinants of ATRX that mediate its PML-NB localization. Various ATRX fragments used for the interaction assays (Fig. 3A) were fused to GFP. When these GFP-ATRX fragment fusions were expressed in mammalian cells, most of them, including those consisting of the first Daxx-interacting domain, showed a diffuse distribution in the nucleus and/or cytosol. However, two ATRX fragments (F1 and F5) appeared in nuclear speckled structures (Fig. 8A). Nevertheless, these two fragments, which were missing the first Daxx-interacting domain (see Fig. 3A), did not localize to the PML-NBs even when they were co-expressed with PML and Daxx (Fig. 8B,  panels d-i; Fig. 8C, panels a-c; and data not shown). Interestingly, the ATRX fragments containing the first Daxx-interacting region (F6, F11, F12, and F14) localized to the PML-NBs when they were co-expressed with Daxx and PML (Fig. 8C,  panels d-o). In contrast, the fragments missing the first Daxxinteracting domain were not localized to the nuclear bodies in the presence of Daxx and PML (Fig. 8C, panels a-c and data not shown). The correlation between the ability to bind to Daxx and the PML-NB localization of the ATRX fragments indicates that Daxx targets ATRX to the nuclear bodies. Finally, we co-expressed GFP-ATRX(F11) with either full-length Daxx or with a Daxx deletion mutant lacking either the NH 2 -terminal ATRX-interacting domain or the COOH-terminal PML-inter-acting domain. The expression of full-length Daxx, but not of the deletion mutants, was correlated with GFP-ATRX accumulation in the PML-NBs. Taken together, these results show that Daxx recruits ATRX to the PML-NBs most likely through its ability to bind to both PML and ATRX. DISCUSSION Daxx was first identified as a regulator of apoptosis through JNK activation. Subsequent studies have shown that it is also a key component of the PML nuclear bodies and that it regulates transcription through its interactions with various DNAbinding proteins. In the present study, we demonstrate that Daxx interacts with ATRX, providing new insights as to the role of Daxx in transcriptional regulation.
Sequence homology has shown that ATRX is a member of the SNF2 family of ATP-dependent chromatin remodeling proteins (23,24). Here we demonstrate that ATRX has nucleosomestimulated ATPase activity that, in the case of purified recombinant ATRX, is equivalent to other recognized chromatin remodeling proteins. In addition, Xue et al. (31) recently showed that an ATRX⅐Daxx complex appeared to have ATP-dependent remodeling activity unique from that of other SWI/SNF-like protein complexes in that it may function at the DNA entry/exit sites of nucleosomes.
Although the ATPase domain of ATRX is a common site of patient mutations (25%), the defects in this domain lie outside of the most highly conserved residues, thereby suggesting that mutations in conserved amino acids may be lethal (23). Consistent with this idea, we demonstrated that two patient mutations (Ped3 and Ped23) introduced into recombinant ATRX attenuated ATPase function, whereas a mutation in the highly conserved catalytic site (K1600R; Mu) totally abolished it. In contrast, a mutation in the PHD domain had normal ATPase activity, suggesting that mutations in this domain have no direct effect on ATPase activity but rather alter ATRX function by another mechanism. Previous studies have indicated that chromatin-remodeling molecules can activate the expression of some genes and repress the expression of others. Interestingly, we found that ATRX, when tethered to DNA, strongly repressed transcription driven by the promoter of the thymidine kinase (Fig. 6). Consistent with this finding is the localization of ATRX to pericentromeric heterochromatin and its interaction with HP-1, both further substantiating a repressive role for ATRX. Moreover, with regard to the present study we expect that the ATPase-dependent chromatin remodeling activity of ATRX is required for transcription repression.
Daxx does not affect the ATPase activity of ATRX. Rather, it appears to regulate the cellular localization of ATRX. We demonstrate that Daxx can target ATRX to the PML-NBs, and, therefore, it may modulate ATRX activity by sequestering ATRX away from its target genes (Fig. 9). It remains to be determined whether Daxx also targets ATRX to sequence-specific transcription factors. This scenario is plausible because Daxx interacts with ATRX through its NH 2 -terminal PAH domains but with sequence-specific transcription factors, such as Pax3, Pax5, ETS1, and the glucocorticoid receptor, through its COOH terminus (17)(18)(19)(20)(21). Daxx may thus function as a molecular bridge between DNA-binding proteins and chromatin remodeling complexes under certain circumstances and facilitate the assembly of transcription regulatory complexes.
The PML-NBs regulate a wide range of cellular processes, including tumor and growth suppression, apoptosis, anti-viral responses, and cellular senescence (11,12). The underlying mechanisms of the PML-NBs, however, are not well defined. Nevertheless, accumulating evidence suggests a role for the PML-NBs in transcriptional regulation (16). Several transcription regulatory proteins have been found in the PML-NBs, including transcription factors such as p53 and pRB, as well as CBP, a member of the histone acetyltransferase protein family. The identification of ATRX, a chromatin-remodeling protein, within the PML-NBs strengthens the notion that these nuclear structures may be critical compartments for the regulation of transcription. It is conceivable that the PML-NBs may regulate transcriptional activity by restricting the access of regulatory proteins to their target genes. Alternatively, it may facilitate the assembly of transcriptional regulatory complexes by bringing the complex components within close proximity of each other.
The function of an ATRX⅐Daxx complex can only be speculated at this point. However, we know that both proteins are required for normal development. Mutations in ATRX lead to a severe mental retardation disorder, whereas mice deficient in Daxx are embryonic lethal. It is thus possible that the ATRX⅐Daxx complex regulates the transcription of developmentally important genes. Confirmation of such a function will require the identification of specific target genes. ATRX⅐Daxx represents a novel transcription regulatory complex with ATRX being the core ATPase/helicase whose localization is regulated by Daxx. Daxx may sequester ATRX away from certain sequence-specific transcription factors (indicated by filled circle) and into the PML-NBs. The ability of Daxx to recruit Daxx into the PML-NBs is likely to be enhanced by PML.

FIG. 8. Daxx targets ATRX to the PML-NBs.
A, cellular localization of various ATRX deletion mutants. GFP-ATRX fusions containing different regions of ATRX were introduced into 293T cells, and the cellular localization of the GFP fusion proteins was examined by epi-fluorescence microscopy. B, the ATRX NH 2 -terminal fragments do not localize to the PML-NBs. 293T cells were transfected with 250 ng of ATRX-GFP fusion plasmids plus 50 ng of RFP-PML. Images were recorded by a confocal microscope. FL, full-length ATRX. C, the first Daxx-interacting region of ATRX mediates its localization to the PML nuclear bodies. The indicated GFP-ATRX expression plasmids were transfected into 293T cells together with PML and Daxx expression plasmids (250 ng of each plasmid). Cells were incubated with anti-PML antibody followed by Texas Red-conjugated anti-mouse IgG antibody. The stained cells were analyzed by confocal microscopy. D, the PML-NB localization of ATRX requires the full-length Daxx protein. GFP-ATRX(F11) (250 ng) was transfected into 293T cells together with the same amount of PML and either Daxx, Daxx-(1-590), or Daxx-(570 -740) plasmids. Cells were incubated with anti-PML antibody followed by Texas Red-conjugated anti-mouse IgG antibody, before being analyzed by confocal microscopy.