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Molecular Endocrinology, doi:10.1210/me.2007-0143
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Molecular Endocrinology 21 (10): 2403-2415
Copyright © 2007 by The Endocrine Society

Glucocorticoid-Induced Degradation of Glycogen Synthase Kinase-3 Protein Is Triggered by Serum- and Glucocorticoid-Induced Protein Kinase and Akt Signaling and Controls ß-Catenin Dynamics and Tight Junction Formation in Mammary Epithelial Tumor Cells

Kim L. Failor, Yelena Desyatnikov, Lindsay A. Finger and Gary L. Firestone

Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California at Berkeley, Berkeley, California 94720-3200

Address all correspondence and requests for reprints to: Gary L. Firestone, Department of Molecular and Cell Biology, 591 Life Sciences Addition, University of California at Berkeley, Berkeley, California 94720-3200. E-mail: glfire{at}berkeley.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoid hormones stimulate adherens junction and tight junction formation in Con8 mammary epithelial tumor cells and induce the production of a stable nonphosphorylated ß-catenin protein localized exclusively to the cell periphery. Glycogen synthase kinase-3 (GSK3) phosphorylation of ß-catenin is known to trigger the degradation of this adherens junction protein, suggesting that steroid-activated cascades may be targeting this protein kinase. We now demonstrate that treatment with the synthetic glucocorticoid dexamethasone induces the ubiquitin-26S proteasome-mediated degradation of GSK3 protein with no change in GSK3 transcript levels. In transfected cells, deletion of the N-terminal nine amino acids or mutation of the serine-9 phosphorylation site on GSK3-ß prevented its glucocorticoid-induced degradation. Expression of stabilized GSK3 mutant proteins ablated the glucocorticoid-induced tight junction sealing and resulted in production of a nonphosphorylated ß-catenin that localizes to both the nucleus and the cell periphery in steroid-treated cells. Serine-9 on GSK3 can be phosphorylated by Sgk (serum- and glucocorticoid-induced protein kinase) and by Akt. Expression of dominant-negative forms of either Sgk- or Akt-inhibited glucocorticoid induced GSK3 ubiquitination and degradation and disrupted the dexamethasone-induced effects on ß-catenin dynamics. Furthermore, the steroid-induced tight junction sealing is attenuated in cells expressing dominant-negative forms of either Sgk or Akt, although the effect of blunting Sgk signaling was significantly greater. Taken together, we have uncovered a new cellular cascade in which Sgk and Akt trigger the glucocorticoid-regulated phosphorylation, ubiquitination, and degradation of GSK3, which alters ß-catenin dynamics, leading to the formation of adherens junctions and tight junction sealing.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
EPITHELIAL CELL POLARITY is determined and maintained by the apical junction complex, which is located at the points of contact between cells and is composed of adherens junctions and tight junctions (1, 2). The tight junction, or zonula occludens, is the most apical structure of the junctional complex and controls the selective diffusion of solutes through a paracellular pathway as well as restricting the lateral diffusion of lipids and membrane proteins between the compositionally distinct apical and basolateral membrane domains (3, 4, 5, 6, 7, 8). The adherens junction, which lies immediately basal to the tight junction, is responsible for intercellular adhesion between neighboring cells (9, 10). Adherens junction formation is required for, and in some systems triggers, the recruitment of tight junction components to the points of cell-cell contact and formation of a tight paracellular seal of the epithelium (11). Adherens junctions and the tight junctions each contain a unique set of transmembrane proteins that form intercellular contacts (12, 13, 14, 15), and tethered to the respective cytoplasmic tails of these transmembrane proteins in a protein complex that comprises peripheral membrane proteins, actin cytoskeleton-binding proteins, and cell signaling molecules (5, 7). The architecture of the apical junction complex, the cytoskeleton, and sealing of tight junctions at the sites of cell-cell contact can be regulated in a dynamic manner depending on the tissue origin, physiological state, and availability of specific sets of extracellular cues (16, 17, 18).

Dramatic molecular rearrangement of the tight junctions occurs in response to hormones in mammary epithelial cells in preparation for lactation (19). Before and during pregnancy, the epithelial lining of the mammary gland is permeable to large molecules, indicating that tight junctions have not yet sealed (20). After parturition, the permeability of the epithelium dramatically decreases due to the sealing of tight junctions between the cells (21). Given that the morphological changes in the mammary gland are triggered by changes in endocrine signaling, it is reasonable to assume that these hormones are triggering a network of signaling cascades that results in dramatic molecular changes in the epithelial cells themselves. Indeed, our work and others have shown that lactogenic hormonal signaling is crucial for the organization and sealing of the apical junctional complex in preparation for lactation, specifically glucocorticoids, one class of steroid hormones (19, 22, 23).

Treatment of cultured mammary epithelial tumor cells with the synthetic glucocorticoid dexamethasone induces adherens junction organization, tight junction sealing, and cell polarity through a multistep cascade (24, 25, 26, 27, 28). An early glucocorticoid-regulated step in this cascade is the rapid stimulation of expression of the helix-loop-helix transcriptional regulatory molecule Id-1, which is required for the steroid control of the apical junction complex (29). In a later set of events, glucocorticoids induce the recruitment of the tight junction proteins (ZO-1 and occludin) and adherens junction proteins (ß-catenin and E-cadherin), as well as the Ras and phosphatidylinositol 3-kinase (PI 3-kinase) cell signaling proteins to the cell periphery at the sites of cell-cell contact (28). This junctional reorganization requires the down-regulation of the RhoA small GTPase (30) and of fascin (26), an actin-bundling protein that binds to the adherens junction protein ß-catenin, as well as the maintenance of the natural RhoA antagonist Rnd3 (31). As part of this process, glucocorticoids stimulate the level of the Rho kinase ROCK2 and down-regulate ROCK1-specific activity (32), both down stream effectors of RhoA. Adherens junction organization is followed by a distinct Ras- and PI 3-kinase-dependent step that results in formation of highly sealed tight junctions in a polarized cell monolayer (28).

A key target of the glucocorticoid-induced signaling cascade in mammary epithelial cells that directly links steroid signaling to components of the apical junction complex is ß-catenin. We have observed that glucocorticoids increase the level of mRNA transcripts and stabilize a nonphosphorylated form of ß-catenin protein that is localized exclusively to the cell periphery. In the absence of steroids, the cytoplasmic ß-catenin is N-terminally phosphorylated and degraded by the 26S proteasome through ubiquitin conjugation. A portion of this phosphorylated ß-catenin localizes to the nucleus and appears to escape being degraded by this process (33). It is well established that ß-catenin is phosphorylated on its N terminus by glycogen synthase kinase-3 (GSK3), which is a process that is regulated canonically through the Wnt signaling pathway (34). Activation of this pathway, involving the Dishevelled protein, results in the inhibition of GSK3 enzymatic activity, thus preventing the N-terminal phosphorylation of ß-catenin (34). The loss of ß-catenin phosphorylation prevents its interaction with the ß-transducin repeat-containing protein (ß-TrCP), E3 ubiquitin ligase, and inhibits the ubiquitin-26S proteasome-mediated degradation of ß-catenin (35).

Several other signaling pathways inhibit GSK3 activity, such as growth factor receptor pathways that activate PI 3-kinase signaling (36). Both Sgk (serum- and glucocorticoid-induced protein kinase) and Akt require PI 3-kinase signaling for their activity, and both of these protein kinases phosphorylate the N terminus of GSK3, serine-21 on GSK3-{alpha}, and serine-9 on the ß-isoform (37, 38, 39). This PI 3-kinase-dependent N-terminal phosphorylation of GSK3 has an autoinhibitory effect, acting as a pseudosubstrate for the kinase (40). Although GSK3 has been shown to be regulated by steroids in human embryonic kidney 293 cells (41), nothing is known about its regulation by glucocorticoids or its functional relationship to adherens junction organization and tight junction sealing. In this study, we show that glucocorticoids induce a novel regulation of GSK3 by causing its modification by ubiquitin and proteasome-mediated degradation in rat mammary epithelial tumor cells. This signal-dependent degradation of GSK3 is triggered by the Sgk- and Akt-dependent phosphorylation of GSK3 and mediates the glucocorticoid control of ß-catenin dynamics and formation of the apical junction complex. These findings represent the first evidence of glucocorticoid regulation of GSK3 and its effects on cell-cell interactions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoids Induce the Ubiquitin-Proteasome-Mediated Degradation of GSK3 Protein
Our previous work demonstrated that treatment of rat Con8 mammary epithelial cells with the synthetic glucocorticoid dexamethasone significantly inhibited the N-terminal phosphorylation of ß-catenin (33), suggesting that glucocorticoids regulate the upstream protein kinase that is responsible for this modification. Because the N terminus of ß-catenin is commonly phosphorylated by glycogen synthase kinase 3-ß (GSK3-ß) in many systems (42), we tested whether GSK3 expression is affected by dexamethasone treatment. Total GSK3 protein and transcript levels were examined in Con8 cells treated with or without 1 µM dexamethasone during a 5-d time course. As shown in Fig. 1AGo, Western blots revealed that the protein levels of both GSK3-{alpha} (upper bands) and GSK3-ß (lower bands) were significantly down-regulated in dexamethasone-treated cells. Maximal reduction of GSK3 protein levels was observed by 120 h of steroid treatment, which is the time point in which this steroid maximally stimulates the protein level and localization of the nonphosphorylated ß-catenin in the adherens junction of these cells (33).


Figure 1
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  		Fig. 1. Expression of GSK3 Protein and mRNA Transcripts in Glucocorticoid-Treated Con8 Mammary Tumor Cells

A, Con8 cells were treated with or without 1 µM dexamethasone for 24, 72, or 120 h. Total cell lysates were electrophoretically fractionated in sodium dodecyl sulfate polyacrylamide gels, and Western blots were probed with the corresponding antibodies for GSK3 or for actin as a loading control. The two GSK3 protein bands are GSK3-{alpha} (upper band) and GSK3-ß (lower band). B, Total RNA was collected from Con8 cells treated with or without 1 µM dexamethasone for 24, 72, or 120 h. RT-PCR was used to detect the levels of mRNA transcripts for GSK3-{alpha}, GSK3-ß, ß-catenin as a steroid-inducible control, or 18S rRNA as a loading control. PCR products were visualized using a 1% agarose gel stained with GelRed. Dex, Dexamethasone.

 
In a parallel time course, the level of GSK3-{alpha} and GSK3-ß transcripts in dexamethasone-treated and untreated cells were examined by RT-PCR analysis. As shown in Fig. 1BGo, neither transcripts for the {alpha}- or ß-isoforms of GSK3 were affected by steroid treatment. As a positive control for dexamethasone treatment, ß-catenin mRNA transcripts were induced under the conditions in which no changes in GSK3 transcript levels occurred. These results suggest that dexamethasone treatment induces a cell signaling pathway that targets GSK3 protein levels without affecting its transcription.

One well characterized pathway to regulate protein levels utilizes the 26S proteasome to degrade proteins that have been conjugated with ubiquitin (43). To determine whether the 26S proteasome is responsible for the dexamethasone-dependent reduction of GSK3 protein levels, Con8 cells were incubated in the presence or in the absence of dexamethasone for 5 d, and in the 6 h before harvesting, the cells were treated with or without 10 µM MG-132, a relatively specific 26S proteasome inhibitor (44), to stabilize any ubiquitinated proteins. The levels of GSK3 and ß-catenin protein were examined by Western blots. As shown in Fig. 2AGo, the dexamethasone-dependent decrease in GSK3 protein was prevented in the presence of MG-132, indicating that steroid treatment triggers the proteasome-mediated degradation of GSK3 protein. The level of ß-catenin protein was used as a control for MG-132 because it is well established that the N-terminal phosphorylation of this protein signals its E3-mediated ubiquitin conjugation and proteasome-dependent degradation (45). As also shown in Fig. 2AGo, MG-132 treatment resulted in an increase in the level of ß-catenin protein in cells not exposed to dexamethasone under conditions in which actin protein levels remain unchanged.


Figure 2
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  		Fig. 2. Dexamethasone-Regulated Degradation of GSK3 Protein Is Prevented in the Presence of the MG132 Proteasome Inhibitor

A, Con8 cells were treated with or without 1 µM dexamethasone for 5 d, and during the last 6 h of treatment, the cells were incubated in the absence or presence of 10 µM of the 26S proteasome inhibitor MG-132. Western blots of electrophoretically fractionated cell lysates were probed for GSK3, ß-catenin, or actin as a loading control. B, Cells were treated with or without 1 µM dexamethasone and with 10 µM MG-132 for 6 h before harvesting the cells. GSK3-ß was immunoprecipitated from total cell extracts using agarose-conjugated anti-GSK3 antibody. Immunoprecipitated (IP) samples were examined by Western blot for the presence of ubiquitin. Indicated molecular weights were determined using a full-range molecular weight rainbow marker. Dex, Dexamethasone.

 
To directly show that glucocorticoids stimulate the ubiquitin conjugation of GSK3 protein, GSK3 was immunoprecipitated from cells treated with or without dexamethasone for 5 d and with MG-132 6 h before collection. Western blots of the immunoprecipitated material were probed with ubiquitin antibodies. As shown in Fig. 2BGo, the immunoprecipitated GSK3-ß (IP: GSK3) shows ubiquitin staining at a molecular mass slightly heavier than GSK3-ß itself (47 kDa). Each ubiquitin protein weighs approximately 8.5 kDa, causing a ladder of ubiquitinated GSK3-ß protein typical of proteins that that are polyubiquitinated to signal their degradation. The control Western blot (Fig. 2BGo, lower panel) shows the 47-kDa GSK3-ß, in addition to the 51-kDa GSK3-{alpha}. These data directly show for the first time that GSK3-ß is ubiquitinated in a glucocorticoid-dependent manner, which leads to the targeted degradation of this protein in steroid-treated cells.

Deletion of the N-Terminal Nine Amino Acids or Mutation of Serine-9 Prevents the Glucocorticoid-Induced Degradation of GSK3-ß
The ubiquitination of many proteins requires a phosphorylated site that is recognized by the corresponding E3 ligase (46). A well-characterized phosphorylation site in GSK3-ß is at serine-9, which inhibits GSK3 enzymatic activity when phosphorylated (37, 38). Two mutations of GSK3-ß were constructed to determine the potential role of serine-9 in the glucocorticoid-induced degradation of this kinase. One construct was a deletion that results in truncation of the N-terminal nine amino acids (GSK3-ß-{Delta}9), and the other mutation was a point mutation that substitutes alanine for serine at position 9 (GSK3-ß-S9A). Expression vectors encoding hemagglutinin (HA)-tagged wild-type GSK3-ß, HA-tagged GSK3-ß-{Delta}9, and HA-tagged GSK3-ß-S9A were stably transfected into Con8 cells. Western blots of total extracts from transfected cells treated with or without dexamethasone for 5 d revealed that glucocorticoids down-regulate exogenous wild-type GSK3-ß protein; however, both the N-terminal-deleted ({Delta}9) and the serine-mutated (S9A) GSK3-ß proteins were stabilized in steroid-treated cells (Fig. 3AGo). Importantly, Western blots using phospho-GSK3-ß antibodies show that the exogenous wild-type GSK3-ß is phosphorylated in a dexamethasone-dependent [phosphorylation of endogenous GSK3-ß is also steroid regulated (data not shown)] whereas, both GSK3-ß-{Delta}9 and GSK3-ß-S9A remained nonphosphorylated in dexamethasone-treated cells because the serine-9 phosphorylation site was ablated (Fig. 3AGo). In addition, nonphosphorylated, stabilized GSK3-ß mutants were not ubiquitinated in the presence or absence of dexamethasone, as opposed to exogenous wild-type GSK3-ß, which was ubiquitinated in a dexamethasone-dependent manner (Fig. 3AGo).


Figure 3
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  		Fig. 3. Effects of Expressing a Serine-9-Mutated or an N-Terminal Nine-Amino Acid-Truncated GSK3-ß on Glucocorticoid-Induced GSK Protein Degradation, ß-Catenin Protein Stability and Phosphorylation, and on GSK3-ß Enzymatic Activity

A, Con8 cells stably expressing exogenous wild-type GSK3-ß, GSK3-ß-{Delta}9, or GSK3-ß-S9A were treated with or without 1 µM dexamethasone for 5 d. Electrophoretically fractionated cell lysates were examined by Western blot with antibodies against GSK3 or actin as a loading control. To examine phosphorylation, exogenous GSK3 was immunoprecipitated from cell lysates with anti-HA antibodies, and the samples were examined by Western blot using antibodies for phosphorylated GSK3. To examine ubiquitination, cells were treated with 1 µM dexamethasone for 5 d and 10 µM MG-132 6 h before harvesting the cell. GSK3 was immunoprecipitated from cell extracts using agarose-conjugated anti-GSK3 antibody, and the immunoprecipitated material was examined by Western blots for the presence of ubiquitin. B, Con8 cells stably expressing exogenous wild-type GSK3-ß, GSK3-ß-{Delta}9, or GSK3-ß-S9A were treated with or without 1 µM dexamethasone for 5 d. Western blot analysis of cell lysates was completed with antibodies against ß-catenin, phosphorylated ß-catenin, or actin as a loading control. C, Con8 cells stably expressing exogenous wild-type GSK3-ß, GSK3-ß-{Delta}9, or GSK3-ß-S9A were treated with or without 1 µM dexamethasone for 5 d. GSK3 kinase activity was measured by incubating immunoprecipitated GSK3 protein with ATP and recombinant GST-ß-catenin protein. Phosphorylated ß-catenin was detected by Western blotting. Dex, Dexamethasone; wt, wild type.

 
The serine-9 phosphorylation inhibits GSK3 enzymatic activity (47), and both GSK3-ß-{Delta}9 and GSK3-ß-S9A are enzymatically active as shown by a kinase assay of immunoprecipitated GSK3 using recombinant ß-catenin protein (Fig. 3CGo). To determine the effects of the mutant forms of GSK3-ß on the stability and phosphorylation of its substrate, ß-catenin, the transfected Con8 cells were treated with or without dexamethasone for 5 d, and the total and phosphorylated ß-catenin protein was examined by Western blot analysis. Consistent with our previous study, in cells expressing wild-type GSK3-ß, dexamethasone stimulated ß-catenin protein levels, which were almost entirely nonphosphorylated (Fig. 3BGo, left lanes). In contrast, in cells expressing GSK3-ß-{Delta}9 or GSK3-ß-S9A, dexamethasone failed to increase total ß-catenin levels, and the residual ß-catenin was phosphorylated (Fig. 3BGo, middle and right lanes). Thus, in Con8 cells forced to express nonphosphorylated active forms of GSK3-ß, ß-catenin is no longer stabilized in a steroid-dependent manner.

Expression of GSK3-ß-S9A Disrupts the Glucocorticoid-Mediated Formation of the Apical Junctional Complex and Tight Junction Sealing
ß-Catenin is a key component of the adherens junction and is necessary for the glucocorticoid-induced formation of adherens junctions and tight junction sealing in Con8 mammary tumor cells (33). We therefore examined whether expression of the stabilized GSK3-ß-S9A, in which ß-catenin remains phosphorylated in steroid-treated cells, has functional consequences on the junctional organization. Con8 cells expressing exogenous wild-type GSK3-ß or GSK3-ß-S9A were treated with or without dexamethasone, and the localization of ß-catenin was examined by indirect immunofluorescence microscopy. In cells expressing wild-type GSK3-ß, ß-catenin was localized primarily to the nucleus in the absence of steroid treatment [compared with the nuclear 4',6-diamidino-2-phenylindole (DAPI) staining for DNA] and was localized exclusively to the cell periphery in dexamethasone-treated cells (Fig. 4AGo, left panels). The same results are observed in nontransfected Con8 cells (33). In contrast, in cells expressing GSK3-ß-S9A, a significant fraction of ß-catenin remained in the nucleus (compared with the nuclear DAPI staining for DNA) in the presence of dexamethasone (Fig. 4AGo, right panels). In the presence of dexamethasone, a portion of ß-catenin did properly localize to the cell periphery. Cells transfected with GSK3-ß-S9A are generally larger than the cells transfected with wild-type GSK3-ß, which accounts for the slight size difference in the nuclei between the two cell populations.


Figure 4
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  		Fig. 4. Disruption of ß-Catenin Localization and Apical Junctional Organization in Cells Stably Transfected with a Stabilized Mutant of GSK3-ß

A, Con8 cells stably expressing exogenous wild-type GSK3-ß or GSK3-ß-S9A were grown to 100% confluency on Nunc permeable supports and treated with or without 1 µM dexamethasone for 5 d. Indirect immunofluorescence microscopy was used to visualize ß-catenin (an adherens junction protein, top panels). DAPI staining was used to visualize DNA (the nucleus, bottom panels). B, Nuclear and nonnuclear fractions were harvested from Con8 cells stably expressing either exogenous wild-type GSK3-ß or GSK3-ß-S9A as described in Materials and Methods. The cells were treated with or without 1 µM dexamethasone for 5 d, and the subcellular fractions were electrophoretically fractionated and examined by Western blots for ß-catenin. Dex, Dexamethasone.

 
To demonstrate the fidelity of the immunofluorescence assay, a subcellullar fractionation was carried out to determine the level of ß-catenin protein in the nuclear and nonnuclear fractions from Con8 cells stably transfected with wild-type GSK3-ß or GSK3-ß-S9A. The nonnuclear fraction corresponds to the supernate of a 14,000 x g centrifugation and includes the cytoplasm and nonnuclear membranes of the cells. As shown in Fig. 4BGo, in cells expressing wild-type GSK3-ß, ß-catenin resides primarily in the nuclear fraction in the absence of steroid treatment, whereas this adherens junction component fractionates primarily with the nonnuclear fraction in dexamethasone-treated cells. In contrast, in cells expressing the GSK3-ß-S9A, a significant fraction of ß-catenin protein remains in the nuclear fraction in dexamethasone-treated cells and resides primarily in the nuclear fraction in the absence of steroid treatment. These subcellular fractionation results confirm our indirect immunofluorescence microscopy data. It is tempting to consider that the portion of ß-catenin that stably resides in the nucleus in dexamethasone-treated GSK3-ß-S9A-expressing cells mediates cell signaling events not normally observed when the cells express wild-type GSK3-ß. The immunofluorescence studies also revealed that the dexamethasone-induced cobblestone staining pattern of the tight junction protein ZO-1 was somewhat disorganized in cells expressing GSK3-ß-S9A compared with cells expressing exogenous wild-type GSK3-ß (data not shown).

Expression of GSK3-ß-S9A disrupted the glucocorticoid-induced organization of adherens and tight junction proteins and promotes the expression of a phosphorylated ß-catenin in glucocorticoid-treated cells. Therefore, the potential effects of expressing this mutant GSK3-ß on tight junction sealing was examined in dexamethasone-treated and untreated cells by monitoring monolayer transepithelial electrical resistance (TER). The electrical resistance across a confluent monolayer of cells is directly proportional to the sealing of the tight junctions between the cells (24). In Con8 cells expressing wild-type GSK3-ß, dexamethasone strongly induced TER of the cell monolayer, whereas during the entire time course of the experiment, the TER remained low in the absence of steroid (Fig. 5Go wt GSK3-ß + Dex vs. wt GSK3-ß – Dex). In contrast, in cells expressing the stabilized GSK3-ß-S9A, the steroid induction of tight junction sealing was significantly blunted. Dexamethasone treatment caused only a minor increase in TER compared with untreated cells (GSK3-S9A + Dex vs. GSK3-S9A – Dex). Taken together, these results implicate the dexamethasone-dependent degradation of GSK3 as a necessary step in the glucocorticoid-regulated organization of the apical junction complex and tight junction sealing, because expressing a stabilized form of this protein kinase disrupts these steroid-regulated processes.


Figure 5
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  		Fig. 5. Disruption of Tight Junction Sealing in Cells Stably Transfected with a Stabilized Mutant of GSK3-ß

Con8 cells stably expressing exogenous wild-type GSK3-ß or GSK3-ß-S9A were grown to 100% confluency on Nunc permeable supports and treated with or without 1 µM dexamethasone for 5 d. TER was measured every 24 h starting at the beginning of the steroid treatment. The results are an average of three independent experiments. Dex, Dexamethasone; wt, wild type.

 
Disruption of Sgk and Akt Signaling Prevents the Glucocorticoid-Induced GSK3 Ubiquitination and Stabilizes GSK3 Protein
The dependence on the serine-9 phosphorylation site of GSK3 for its glucocorticoid-induced degradation and the stimulation of adherens junction and tight junction formation suggests that key upstream effectors of GSK3 target this site and may be under steroid control. Two potential protein kinases that are known to phosphorylate GSK at serine-9 are Sgk and Akt. Sgk expression is strongly stimulated by glucocorticoids by glucocorticoid receptor binding a glucocorticoid response element in the Sgk promoter (48, 49). In addition, both Sgk and Akt are activated in a PI 3-kinase-dependent manner, and we have previously shown that PI 3-kinase signaling is critical for the glucocorticoid control of adherens junction and tight junction formation (28). Therefore, to test the role of Sgk and Akt in controlling the glucocorticoid regulation of GSK3 protein, Con8 cells were stably transfected with wild-type and dominant-negative (DN) forms of the protein kinases (DN-Sgk and DN-Akt). The dominant negative Sgk is formed using a double mutation in which the phosphorylation sites at threonine 256 and serine 421 are converted to alanines (50) and the DN-Akt is formed by the conversion of ATP binding site at lysine 179 to alanine (51). The DN properties of both Sgk and Akt were previously characterized (50, 51). The effects on ubiquitination, degradation, and phosphorylation of GSK3 were examined in transfected cells treated with or without dexamethasone for 5 d. The levels of phosphorylated and total GSK3 protein were examined in Western blots using specific antibodies. For the analysis of GSK3 ubiquitination, cells were treated with dexamethasone for 5 d and 10 µM MG-132 6 h before harvesting the cells. Immunoprecipitated GSK3 was electrophoretically fractionated and the Western blot was probed for the presence of ubiquitin.

Western blots revealed that expression of DN-Sgk and DN-Akt prevented the dexamethasone-induced degradation and ubiquitination of GSK3, and that the expressed GSK3 protein was not phosphorylated (Fig. 6Go). In cells expressing either wild-type Sgk or wild-type Akt, dexamethasone strongly induced GSK3 ubiquitination, and degradation, and in steroid-treated cells there were higher levels of the residual phosphorylated form of GSK3 (Fig. 6Go). Similar results were observed in nontransfected Con8 cells (see Figs. 1Go and 2Go) or in vector-transfected control cells (data not shown). These data show that Sgk and Akt signaling by the active forms of these protein kinases is required for the glucocorticoid-induced ubiquitination and degradation of GSK3, implicating the Sgk- and Akt-mediated phosphorylation of serine-9 of GSK3 in controlling GSK3 protein stability.


Figure 6
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  		Fig. 6. Effects of Expressing DN and Wild-Type Forms of Sgk or Akt on the Glucocorticoid Regulation of GSK3 Protein Degradation, Ubiquitination, and Phosphorylation

Cells stably expressing exogenous wild-type or DN-Sgk or -Akt were treated with or without 1 µM dexamethasone for 5 d. Electrophoretically fractionated total cell lysates were examined by Western blot with antibodies against GSK3, phospho-GSK3, or actin as a loading control. To examine ubiquitination, cells were treated with or without 1 µM dexamethasone for 5 d and 10 µM MG-132 6 h before harvesting the cells. GSK3 was immunoprecipitated for total cell lysates using agarose-conjugated anti-GSK3 antibody, and the production of ubiquitinated GSK3 was examined by Western blot analysis for the presence of ubiquitin. Dex, Dexamethasone; wt, wild type.

 
Expression of DN Forms of Sgk and Akt Disrupts the Glucocorticoid-Induced Stabilization of Nonphosphorylated ß-Catenin
Expression of GSK3-ß-S9A, which lacks its Sgk and Akt phosphorylation site, inhibited the glucocorticoid-induced stabilization of nonphosphorylated ß-catenin (see Fig. 3BGo), and therefore disruption of upstream signaling components that alter the glucocorticoid-induced degradation of GSK3 protein would be predicted to have a similar effect on ß-catenin protein. To test this possibility, total and N-terminally phosphorylated ß-catenin levels were examined in cells expressing wild-type or DN forms of Sgk and Akt. Transfected cells were treated with or without dexamethasone for 5 d, and Western blots were probed with specific ß-catenin and phospho-ß-catenin antibodies. As shown in Fig. 7Go, steroid treatment of cells expressing exogenous wild-type Sgk and Akt resulted in an increase in total ß-catenin protein levels that is not recognized by the phospho-ß-catenin antibodies. Similar results were observed in nontransfected cells or in control vector-transfected Con8 cells (data not shown). Importantly, in cells expressing DN-Sgk or DN-Akt, glucocorticoids failed to up-regulate ß-catenin, and the resulting protein was phosphorylated in the presence or absence of steroid (Fig. 7Go). Thus, expression of DN forms of Sgk or Akt, both upstream effectors for GSK3 degradation, has the same effect on ß-catenin protein as does expression of a stabilized form of GSK3-ß that is mutated in its Sgk/Akt phosphorylation site at serine-9.


Figure 7
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  		Fig. 7. Effects of Expressing DN and Wild-Type Forms of Sgk or Akt on the Glucocorticoid Control of Total and Phosphorylated ß-Catenin Protein Levels

Con8 cells stably expressing exogenous wild-type or DN-Sgk or -Akt were treated with or without 1 µM dexamethasone for 5 d. Western blot analysis of electrophoretically fractionated total cell lysates was completed with antibodies against ß-catenin, phosphorylated ß-catenin, or actin as a loading control. Dex, Dexamthasone; wt, wild type.

 
Expression of DN Forms of Sgk or Akt Alters the Glucocorticoid-Regulated Localization of ß-Catenin and Disrupts Steroid-Induced Tight Junction Sealing
The effect on ß-catenin localization was examined by indirect immunofluorescence in Con8 cells expressing DN forms of Sgk or Akt treated with or without dexamethasone. In cells expressing wild-type Sgk or Akt, dexamethasone treatment shifted the subcellular localization of ß-catenin from nuclear to almost exclusively residing at the cell periphery (Fig. 8Go, left panels). This result is similar to that observed with nontransfected cells (33) or cells transfected with wild-type GSK3-ß (see Fig. 4Go). In contrast, expression of either DN-Sgk or DN-Akt altered the dexamethasone-induced localization of ß-catenin. In these transfected cells, a significant fraction of ß-catenin remained nuclear in the presence of dexamethasone, as monitored by DAPI costaining for DNA (Fig. 8Go, right panels). The overall pattern of ß-catenin staining in cells expressing DN-Sgk or DN-Akt was generally similar to that of cells expressing GSK3-ß-S9A in that the same fraction of ß-catenin resides in the nucleus or the cell periphery in the presence of glucocorticoids. Expression of either DN-Sgk or DN-Akt also altered the glucocorticoid-regulated localization of the ZO-1 tight junction protein (data not shown) in a manner similar to that observed in the presence of the stabilized GSK3-ß-S9A.


Figure 8
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  		Fig. 8. Effects of Expressing DN and Wild-Type Forms of Sgk or Akt on the Localization of ß-Catenin

Con8 cells stably expressing exogenous wild-type or DN-Sgk (A) or -Akt (B) were grown to 100% confluency on Nunc permeable supports and treated with or without 1 µM dexamethasone for 5 d. Indirect immunofluorescence microscopy was used to visualize ß-catenin (top panels). DAPI staining was used to visualize DNA (bottom panels). Dex, Dexamethasone; wt, wild type.

 
The effects of disrupting Sgk and Akt signaling on the glucocorticoid-induced tight junction sealing was also assessed using the TER assay. In cells expressing exogenous wild-type Sgk or wild-type Akt, the electrical resistance of the cell monolayers significantly increased in response to dexamethasone treatment over a 6-d time course, whereas, in the absence of steroids, there were no changes in tight junction sealing (Fig. 9Go, A and B). These results were nearly identical to what we have reported for nontransfected Con8 cells (33). In contrast, expression of DN-Sgk strongly disrupted the kinetics and magnitude of the glucocorticoid-induced tight junction sealing (Fig. 9AGo, DN-Sgk + Dex). In cells expressing DN-Akt there was a delayed and reduced increase in electrical resistance in response to dexamethasone treatment (Fig. 9BGo, DN-Akt + Dex). Under these conditions, dexamethasone induces the expression of endogenous Sgk, which may explain why the effects of DN-Akt are less disruptive compared with DN-Sgk. In the absence of glucocorticoids, neither DN protein kinase had any effect on the basal electrical resistance.


Figure 9
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  		Fig. 9. Effects of Expressing DN and Wild-Type Forms of Sgk or Akt on the Glucocorticoid-Induced Tight Junction Sealing

Con8 cells stably expressing exogenous wild-type or DN-Sgk (A) or -Akt (B) were grown to 100% confluency on Nunc permeable supports and treated with or without 1 µM dexamethasone for 5 d. TER was measured every 24 h starting at the beginning of treatment. The results are an average of three independent experiments. The average error was 12.7 ohms x cm2 for wild-type Sgk-Dex samples, 11.8 ohms x cm2 for DN-Sgk-Dex, 3.1 ohms x cm2 for wild-type Akt -Dex, and 5.2 ohms x cm2 for DN-Akt-Dex samples. Dex, Dexamethasone; wt, wild type.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Our previous studies showed that glucocorticoids induce a stable nonphosphorylated ß-catenin that resides exclusively at the cell periphery. This cellular process is part of a steroid-regulated pathway that stimulates adherens junction formation and tight junction sealing. Our new results have uncovered the upstream components of the glucocorticoid-regulated cascade that targets ß-catenin in mammary epithelial tumor cells and that controls cell-cell interactions. As illustrated in Fig. 10Go, we propose that glucocorticoids induce the Sgk- and Akt-mediated N-terminal phosphorylation of GSK3, which causes the ubiquitination and degradation of both GSK3 isoforms. Because the GSK3 phosphorylation of ß-catenin signals the subsequent ubiquitination and degradation of ß-catenin (52), the loss of GSK3 protein explains the stable accumulation of nonphosphorylated ß-catenin that is accessible to the adherens junction at the cell periphery. In the absence of glucocorticoids, ß-catenin remains nuclear and thereby inaccessible to its proteasome-mediated degradation. The nonphosphorylated ß-catenin that accumulates in the nucleus in other systems has been shown to associate with transcription factors to stimulate expression of target genes such as c-myc, c-jun, and cyclin D1 (53). Our evidence has shown that the serine-9 phosphorylation site in GSK3-ß is a key structural requirement for the steroid-induced degradation of GSK3. Mutation of serine-9 to alanine prevents the ubiquitination and degradation of GSK3-ß and results in expression of a stable form of GSK3-ß that is enzymatically active. Importantly, in glucocorticoid-treated cells, expressing GSK3-ß-S9A, a phosphorylated form of ß-catenin is produced that displays an altered subcellular localization in which a significant portion of ß-catenin remains in the nucleus instead of being localized exclusively to the cell periphery. Under these conditions, the adherens junction remains partially disorganized, and the steroid-induced tight junction sealing is disrupted.


Figure 10
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  		Fig. 10. Proposed Model for the Glucocorticoid Control of Cell-Cell Interactions and ß-Catenin Dynamics through the Regulated Degradation of GSK3 Protein

In the absence of glucocorticoids, the cytoplasmic form of ß-catenin is phosphorylated by GSK3, which initiates the ubiquitination and 26S proteasome-mediated degradation of ß-catenin. The nuclear form of ß-catenin is not degraded. We propose that the glucocorticoid stimulation of Sgk expression and activation, in combination with activated Akt, phosphorylates GSK3 to inactivate this kinase and to trigger its ubiquitination by a specific E3 ubiquitin ligase complex and subsequent 26S proteasome-mediated degradation. As a result of the steroid-induced degradation of GSK3, a stabilized hypophosphorylated form of ß-catenin is produced in glucocorticoid-treated cells that is accessible to be localized to its site of function in the adherens junction and subsequent sealing of the tight junctions. The control of ß-catenin protein stability through the regulation of GSK3 degradation provides a direct molecular link between steroid hormone signaling and the control of cell-cell interactions. Ub, Ubiquitin.

 
Expression of DN forms of either Sgk or Akt, which both can phosphorylate GSK3-ß at serine-9, results in the accumulation of a nonphosphorylated GSK3 that fails to be ubiquitinated or degraded in the presence of glucocorticoids. In these cells, the glucocorticoid control of ß-catenin dynamics, adherens junction organization, and tight junction sealing are either disrupted or attenuated. The likely trigger for the steroid-regulated phosphorylation and degradation of GSK3 is the glucocorticoid-induced expression of Sgk, which we have shown is directly transcriptionally regulated due to the presence of a glucocorticoid response element in its promoter (48, 49). Expression of DN-Sgk has a significantly more disruptive effect on steroid-induced tight junction sealing compared with that observed in the presence of DN-Akt. Although the catalytic domains of Sgk and Akt are homologous, Sgk lacks the pleckstrin homology domain that is present in Akt. Several studies suggest that Sgk and Akt have distinct cellular effects despite their apparent similarities in substrate specificity. For example, a common target of both Sgk and Akt is the forkhead transcription factor in which Sgk and Akt have been proposed to play distinct and overlapping functional roles in controlling forkhead function (54, 55). Also, in 3T3-Li adipoctyes, Akt but not Sgk, stimulates glucose transport activity and oncogenic transformation (39). Conceivably, in glucocorticoid-treated cells, the phosphorylation of GSK3 by a combination of Sgk and Akt ensures the efficient targeting of GSK3 for ubiquitination and degradation. Alternatively, perhaps Sgk and Akt have distinct roles in GSK3 phosphorylation, depending on the overall network of extracellular signals and intracellular pathways that are activated in a physiologically appropriate manner. We have previously observed that glucocorticoids regulate the localization of PI 3-kinase (28), an upstream regulator for both Sgk and Akt (50, 56), suggesting that steroid hormones can control the function of both Sgk and Akt, although the relative contributions of individual growth factor-signaling pathways on Sgk and Akt function are not well established.

A key structural requirement for ubiquitination and subsequent degradation of a variety of proteins is the phosphorylation at a specific site for recognition by the corresponding E3 ubiquitin ligase and subsequent ubiquitination at a nearby site on the protein. One notable example is GSK3 phosphorylation of ß-catenin that generates a recognition site for the ß-TrCP E3 ligase. ß-Catenin is phosphorylated on serine-45 by casein kinase-1, and this phosphorylation acts as a primer for phosphorylation by GSK3 on serines-33, serine-37, and/or threonine-41. ß-Catenin is then ubiquitinated at lysine-19 (57), which is in relatively close proximity to the phosphorylation sites. ß-TrCP has been shown to reside in both the nucleus and the cytoplasmic compartments (58), although there appears to be a compartment-specific function in that the ß-TrCP-dependent degradation of NF-{kappa}B2 p100 occurs exclusively in the cytoplasm (59). Because we observed a nuclear form of phosphorylated ß-catenin in the absence of steroid treatment, it is possible that in steroid-treated cells, the ß-TrCP E3 ligase recognizes or is accessible to ß-catenin only in the cytoplasmic compartment. In this regard, ß-TrCP is a component in a large protein complex and therefore may be selectively functional in the cytoplasmic compartment, and so the nuclear form of ß-catenin escapes the degradative process. The precise E3 ubiquitin ligase that acts on phosphorylated GSK3 is unknown, because our report is the first one showing that GSK3 can be ubiquitinated and degraded in a signal-dependent manner.

The glucocorticoid regulation of GSK3 protein degradation and subsequent consequences on ß-catenin protein dynamics appears to be a highly coupled process that controls cell-cell interactions of mammary epithelial tumor cells. In the Wnt signaling pathway, Axin and anaphase promoting complex serve as a scaffold that binds both ß-catenin and GSK3, forming a multiprotein destruction complex that facilitates phosphorylation of ß-catenin by GSK3, which triggers degradation of ß-catenin (53). Conceivably, GSK3 and ß-catenin reside in the same protein complex in mammary epithelial tumor cells, thus ensuring an efficient interaction between GSK3 and ß-catenin.

Our results suggest that the glucocorticoid control of GSK3 degradation may function to deliver a stable form of ß-catenin to the cell periphery to maintain apical junction organization and tight junction sealing in a manner similar to that observed in normal mammary epithelia. In this regard, ß-catenin is important for the development and differentiation of the mouse mammary gland stimulated by hormones during pregnancy (60). Although mutations of GSK3 have not been observed in human tumors (61), disruption of the GSK3 phosphorylation of ß-catenin has been observed in a variety of cancer cell types. For example, missense mutations of ß-catenin at the GSK3 phosphorylation sites have been found in various human tumors (62), and expression of N-terminally mutated ß-catenin, which is missing its GSK3 phosphorylation site, causes tumor formation in mouse mammary glands (60, 63).

GSK3 signaling has been shown to play a role in a variety of physiological processes and in vertebrate development (47, 64), in part through its role as a key component in the Wnt signaling pathway (34). Alterations in GSK3 enzymatic activity and production have been associated with several disease states including Alzheimer’s disease, mood disorders, and type 2 diabetes (65, 66, 67, 68, 69). Our evidence has shown that glucocorticoid-induced Sgk triggers the steroid-regulated phosphorylation and degradation of GSK3 in mammary epithelial tumor cells, suggesting that this regulatory pathway functions in many types of tumorigenic and normal cell types. Sgk transcription is stimulated by a variety of extracellular signals, and one recent study has shown that the mineralocorticoid stimulation of Sgk causes the phosphorylation and inactivation of GSK3 in human embryonic kidney 293 cells (41). Conceivably, the signal-dependent control of GSK3 phosphorylation and degradation, either by Sgk or Akt, may prove to be an important regulatory switch in mammalian epithelia that controls many physiological cascades including those that control cell-cell interactions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
DMEM/F-12 (50:50) and calf serum were supplied by Cambrex (Walkersville, MD). Dexamethasone and mouse monoclonal antiphospho-ß-catenin (Ser33/37) antibodies were obtained from Sigma Chemical Co. (St. Louis, MO). MG-132 was purchased from EMD Biosciences (San Diego, CA). Mouse monoclonal anti-ß-catenin antibodies were purchased from Zymed Laboratories, Inc. (South San Francisco, CA). Mouse monoclonal anti-GSK3-{alpha}/ß and goat polyclonal antiactin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal antiphospho-GSK3-{alpha}/ß (Ser21/9) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Mouse monoclonal antiubiquitin antibodies were obtained from Novus Biologicals (Littleton, CO). Mouse monoclonal anti-HA antibodies were purchased from Covance Laboratories, Inc. (Berkeley, CA). Texas red-X-conjugated antimouse IgG antibodies were supplied by Molecular Probes, Inc. (Eugene, OR). Oligonucleotide primers for RT-PCR were purchased from Integrated DNA Technologies (Coralville, IA).

Cell Culture and Measurement of Transepithelial Electrical Resistance
Con8 rat mammary epithelial tumor cells were routinely grown to 100% confluency on Nunc permeable supports or Nunc tissue culture plates (Fisher Scientific, Santa Clara, CA) in DMEM/F-12 supplemented with 10% calf serum and penicillin/streptomycin and maintained at 37 C in a humid atmosphere of air/CO2 (95:5). These cells were cultured in serum free medium for 24 h before and during all experiments, and the cell culture medium was routinely changed every 24 h. Cells were treated with or without the synthetic glucocorticoid agonist, dexamethasone, at a final concentration of 1 µM (prepared as 1 mM stock in ethanol). The formation of tight junction was monitored by measuring transepithelial electrical resistance (TER), using an EVOM Epithelial Voltohmmeter (World Precision Instruments, Sarasota, FL) as previously described (24). Calculations for ohms x cm2 were determined by multiplying by the area of the monolayer (0.49 cm2 for the 10-mm filters).

Western Blotting
For Western blot analyses, treated cells were rinsed with PBS and extracted in lysis buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 20 mM MgCl2; and 1% Nonidet P40) containing protease inhibitors (2 µg/ml aprotinin, 1 mM dithiothreitol, 10 mM ß-glycerophosphate, 2 µg/ml leupeptin, 1 µg/ml pepstatin, 0.2 µM phenylmethylsulfonyl fluoride, 1 mM sodium fluoride, and 1 mM sodium orthovanadate). Samples were normalized for protein content with the Bio-Rad DC protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Protein samples were fractionated on a 7.5% sodium dodecyl sulfate-polyacrylamide gel along with a full-range molecular weight rainbow marker (Amersham-GE Healthcare, Arlington Heights, IL). Fractionated proteins were then electrophoretically transferred to a nitrocellulose membrane (Micron Separations, Inc., Westborough, MA). Blots were blocked with blocking buffer (5% nonfat dry milk; 200 mM Tris, pH 8.0; 300 mM NaCl; 0.05% Tween 20) before probing with a 1:1000 dilution of primary antibodies. Horseradish peroxidase-conjugated antirabbit, antimouse, and antigoat antibodies (Bio-Rad) were used as secondary probes, and the blots were developed with Western Lightning enhanced chemiluminescence (PerkinElmer Life Sciences, Boston, MA).

RT-PCR
Total RNA from Con8 cells treated with or without 1 µM dexamethasone was isolated with TRI Reagent according to manufacturer’s protocol (Sigma). Total RNA (2 µg) was used to synthesize cDNA using Moloney murine leukemia virus-reverse transcriptase (Promega Corp., Madison, WI) with random hexamers as primers. the cDNA reaction product (100 ng) was used with 10 µM primers. (GSK3-{alpha}: forward primer, 5'-ACCTACCCTCACTAACTCTTCCTG-3'; reverse primer, 5'-GTAGAAGGTCCTCATACCCCAAAC-3'; GSK3-ß: forward primer, 5'-AGAGAACGAAGTCTTTTTTT-3'; reverse primer, 5'-TTTATCTCTGCTAACTTTCA-3'; ß-catenin: forward primer, 5'-GATTAACTATCAGGATGACGCG-3'; reverse primer, 5'-TCCATCCCTTCCTGCTTAGTC-3') As a loading control, 18S rRNA was amplified from the same samples using a primer/competimer pair (Ambion, Austin, TX). PCR products were analyzed on 1.0% agarose gel stained with GelRed (Biotium, Hayward, CA).

Immunoprecipitation
After treatment, cells were lysed in immunoprecipitation buffer (50 mM Tris, pH 7.4; 250 mM NaCl; 0.1% Triton X-100; 2 µg/ml aprotinin; 10 mM ß-glycerophosphate; 2 µg/ml leupeptin; 1 µg/ml pepstatin; 0.2 µM phenylmethylsulfonyl fluoride; 1 mM sodium fluoride; and 1 mM sodium orthovanadate). Protein was normalized using the Bio-Rad DC Protein Assay. Protein lysate (500 µg) was precleared for 30 min at 4 C with empty Protein G Sepharose (GE Healthcare Biosciences, Arlington Heights, IL). Precleared samples were incubated with 10 µl of agarose-conjugated anti-GSK3 antibody (Santa Cruz Biotechnology) or 10 µl of anti-HA antibody overnight at 4 C. For immunoprecipitation of HA-conjugated proteins, samples were then incubated with Protein G Sepharose beads for 1 h at 4 C. For all immunoprecipitations, beads were gently spun down at 2000 rpm for 2 min and washed three times with immunoprecipitation buffer. Immunoprecipitated protein was eluted from beads with 40 µl of 100 mM glycine at pH 3.0, and samples were analyzed by Western blot as described above using mouse antiubiquitin antibodies (Novus Biologicals, Littleton, CO).

Site-Directed Mutagenesis and Generation of Stably Transfected Cells
Wild-type GSK3-ß and GSK3-ß-{Delta}9 constructs were obtained from the laboratory of David G. Menter. Serine-9 of wild-type GSK3-ß construct was mutated to an alanine to generate the GSK3-ß-S9A mutant construct using site-directed mutagenesis. Template DNA (5 ng) was mixed with 20 pmol of each primer (GSK3-ß-S9A: forward primer, 5'-ACCACCGCCTTTGCGGAGAGCTG-3'; GSK3-ß-S9A reverse primer, 5'-CCGCAAAGGCGGTGGTTCTCGG-3'), and the PCR was run (94 C, 30 sec; 55 C, 1 min; 68 C, 5 min; 12 cycles). The product was then digested with Dpn1 and transformed into TOP10 competent Escherichia coli.

The DN-Sgk construct was generated in a similar way using wild-type Sgk as the template DNA and mutating threonine-256 to alanine and serine-421 to alanine, successively, using the following primers. Sgk-T256A: forward primer, 5'-ACGTCCGCCTTCTGTGGCACGC-3'; reverse primer, 5'-CACAGAAGGCGGACGTTGTCCC-3'; Sgk-S421A: forward primer, 5'-GGCTTCGCCTATGCCCCTCCTATG-3'; reverse primer, 5'-GGGCATAGGCGAAGCCAAGGAAG-3'.

To generate stably transfected cells, Con8 cells were transfected with 0.2 µg of pCMV5neo as a selectable marker and 1.0 µg of wild-type GSK3-ß, GSK3-ß-{Delta}9, GSK3-ß-S9A, wild-type Akt, Akt-K179A (DN-Akt, generously provided by the laboratory of Hei Sook Sul), wild-type Sgk, or Sgk-T256A,S421A (DN-Sgk), using Lipofectamine (Invitrogen, Carlsbad, CA) and the manufacturer’s suggested protocol. cells were fed 24 h after transfection with DMEM/F-12, supplemented with 10% calf serum, penicillin/streptomycin, and 0.7 mg/ml G418 (neomycin analog, Mediatech, Herndon, VA) to select for transfected cells.

GSK3 Kinase Assay
Stably transfected Con8 cells were treated with or without 1 µM dexamethasone for 5 d in serum-free media. The GSK3 kinase assay was performed as described elsewhere (32) with some modifications. GSK3 was immunoprecipitated using the technique described above. After wash steps, beads were resuspended in kinase assay buffer (50 mM HEPES, pH 7.4; 150 mM NaCl; 1 mM MgCl2; 1 mM MnCl2; 5% glycerol) and incubated with 10 µM ATP and 1 µg recombinant GST-ß-catenin (Upstate Biotechnology, Inc., Lake Placid, NY) in a reaction volume of 50 µl for 30 min at 37 C. The reaction was stopped by the addition of SDS-PAGE buffer, followed by incubation at 100 C for 10 min. Kinase activity was detected by Western blotting, using antiphospho-ß-catenin antibodies as described above.

Indirect Immunofluorescence Microscopy
For indirect immunofluorescence assays, cells were grown on Nunc filters. The cells were fixed with 3.5% formaldehyde in PBS for 15 min at room temperature. After three additional washes with PBS, the plasma membrane was permeabilized with Triton X-100 extraction buffer (0.5% Triton X-100; 10 mM Tris-HCl, pH 7.5; 120 mM NaCl; 25 mM KCl; 2 mM EGTA; and 2 mM EDTA) for 10 min at room temperature. Filters were incubated with 3% nonfat dry milk in PBS before incubation with primary antibodies. Mouse anti-ß-catenin antibodies (Zymed Laboratories) were used at a 1:400 dilution. Secondary Texas red-conjugated antimouse antibodies (Molecular Probes, Inc., Eugene, OR) were used at a 1:150 dilution. Stained cells were mounted with Vectashield Mounting media (Vector Laboratories, Inc., Burlingame, CA). Stained and mounted cells were then processed with a Zeiss Axioplan epifluorescence microscope (Carl Zeiss, Thornwood, NY).

Subcellular Fractionation
The nuclear and nonnuclear subcellular fractions were harvested from cell extracts using the Active Motif nuclear extract kit (Active Motif, Carlsbad, CA). Briefly, cells were collected and lysed using a hypotonic solution. Fractions were separated by high-speed centrifugation at 14,000 x g for 30 sec as indicated by the manufacturer’s protocol. Cell fractions were examined by Western blots as described above.


   ACKNOWLEDGMENTS
 
We thank Matt Sweeney, Christine Brew, and Ida Aronchik for their helpful advice and assistance. We thank Kai Li for her technical assistance.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant DK-42799 (to G.L.F.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 26, 2007

Abbreviations: DAPI, 4',6-Diamidino-2-phenylindole; DN, dominant-negative; GSK3, glycogen synthase kinase-3; HA, hemagglutinin; PI 3-kinase, phosphatidylinositol 3-kinase; Sgk, serum- and glucocorticoid-induced protein kinase; TER, transepithelial electrical resistance; ß-TrCP, ß-transducin repeat-containing protein.

Received for publication March 15, 2007. Accepted for publication June 18, 2007.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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