STAT5

Glucocorticoid Receptor/Signal Transducer and Activator of Transcription 5 (STAT5) Interactions Enhance STAT5 Activation by Prolonging STAT5 DNA Binding and Tyrosine Phosphorylation

Shannon L. Wyszomierski, Juddi Yeh, and Jeffrey M. Rosen Department of Cell Biology (S.L.W., J.M.R.) Baylor College of Medicine Houston Texas 77030-3498 University of Texas Health Science Center (J.Y.) San Antonio, Texas 78284

hibited by STAT5 and GR at the b-casein promoter and is an example of how transcription factors at a composite response element may modulate each other’s activity. (Molecular Endocrinology 13: 330– 343, 1999)

The regulation of casein gene expression by both PRL and glucocorticoids has been a well studied paradigm for understanding how the signaling pathways regulated by these two hormones interact in the nucleus. Previous studies have demonstrated that the downstream effectors of these pathways, signal transducer and activator of transcription 5 (STAT5) and the glucocorticoid receptor (GR), are associated via protein-protein interactions and act synergistically to enhance b-casein gene transcription. Indirect immunofluorescence microscopy was used to demonstrate that PRLactivated STAT5 can translocate GR into the nucleus, and that ligand-bound GR can translocate STAT5 into the nucleus. This provided further support of an interaction between the two proteins. To better understand the mechanism of transcriptional synergy between STAT5 and GR, experiments were performed in cells transiently transfected with STAT5 alone or with STAT5 and GR. GR cotransfection enhanced the DNA-binding activity of STAT5 without affecting STAT5 protein levels. The enhancement of STAT5 DNA binding by GR resulted in the formation of a complex that exhibited prolonged DNA binding after PRL treatment. This was correlated with increased STAT5 tyrosine phosphorylation, suggesting that GR enhances STAT5 DNA binding by modulating the rate of STAT5 dephosphorylation. In contrast, cotransfection of the estrogen receptor resulted in an overall decrease in STAT5 tyrosine phosphorylation, without changing the kinetics of dephosphorylation. Enhancement of STAT5 activity by GR is, therefore, one component of the transcriptional synergy ex-

INTRODUCTION The regulation of casein gene expression by both peptide and steroid hormones has been a well studied paradigm for understanding the mechanisms by which the signaling pathways regulated by these two classes of hormones interact in the nucleus. PRL and hydrocortisone (HC) act synergistically to induce b-casein gene expression in mammary epithelial cells (reviewed in Ref. 1). Recently, it has been demonstrated that STAT5 (signal transducer and activator of transcription 5) and the glucocorticoid receptor (GR), the downstream effectors of PRL and HC, respectively, interact and exhibit transcriptional synergy (2). This observation has raised new questions concerning how these two very different signaling pathways impinge on the b-casein promoter. After HC uptake into cells, it binds to intracellular GR. Ligand binding induces a conformational change in GR and dissociation of heat shock proteins, which are associated with the latent form of the receptor (reviewed in Ref. 3). Unlike many steroid receptors, GR resides primarily in the cytoplasm in the absence of ligand. Upon ligand binding, it translocates to the nucleus (4), where it binds to palindromic glucocorticoid response elements (GREs) in the promoters of target genes, as well as interacts with GRE half-sites and other trans-acting factors (reviewed in Refs. 5 and 6). PRL binds to the extracellular portion of the PRL receptor (PRL-R) and initiates events in the JAK/STAT

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signal transduction cascade (7). Specifically, JAK2, which is associated with the PRL-R in the absence of ligand, is activated by transphosphorylation when two JAK2 molecules are brought together by ligandinduced dimerization of the receptor (8). The activated JAK2 tyrosine phosphorylates PRL-R, creating docking sites for SH2 domain-containing proteins, including STAT5 (9), which subsequently are tyrosine phosphorylated, dimerize, and then translocate to the nucleus. Activated STAT5 binds to DNA sites known as GAS elements (named for g-interferon-activated sequences) and modulates the activity of target genes containing GAS elements in their promoters, such as the b-casein gene (10, 11). STAT5 was first identified as a binding activity in tissue extracts from lactating mammary gland and was referred to as MGF (mammary gland factor) (12–14). Molecular cloning revealed that MGF was in fact STAT5 (11) and was neither lactation specific nor mammary gland specific (11, 15, 16). In fact, STAT5 was activated by many hormones, growth factors, and cytokines, including PRL (11, 17–23). Two clustered STAT5 genes, STAT5a and STAT5b, have been identified that are more than 90% identical and probably arose by gene duplication (16). Both STAT5a and STAT5b encode different isoforms, some of which may arise by alternative splicing. In particular, carboxy-truncated isoforms are common for the STAT family of transcription factors (15, 24–28). A carboxytruncated STAT5a isoform, designated STAT5a2, was first identified while screening a cDNA library prepared from RNA isolated from the rat mammary gland at day 2 of lactation. The mRNA for STAT5a2 is generated by a 1.7-kb insertion that encodes a stop codon resulting in a 53-amino acid deletion (15). A very similar alternative splice form, called STAT5bD40C, was identified for STAT5b. STAT5bD40C is truncated at the same position in the protein; however, only 40 amino acids are deleted due to differences in the carboxy-terminal sequences of STAT5a and STAT5b (25). These naturally occurring carboxy-truncated STAT5 isoforms may act as dominant negative inhibitors of STAT5dependent transcription and cannot independently activate transcription because they lack the carboxyterminal transactivation domain. They remain tyrosine phosphorylated and bound to GAS sites for longer periods of time than full-length STAT5 isoforms after PRL treatment, suggesting that the carboxy-terminal sequences may affect the interaction with a tyrosine phosphatase (28, 29). STAT5 and GR have been shown to interact, both in transiently transfected COS cells (2) and in mammary epithelial cells (30). In the HC11 mammary epithelial cell line, STAT5 and GR are associated independently of HC and PRL treatment. This association was also observed in tissue extracts prepared at all stages of mammary gland development (30). STAT5 and GR activate transcription from the b-casein promoter in a synergistic fashion in transiently transfected COS cells (2), and both STAT5a and STAT5b synergize with GR

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(31). The C-terminal transactivation domain of STAT5 is not necessary for this transcriptional synergy (31, 32), but the amino-terminal TAF-1 domain of GR is required (31). One mechanism proposed for STAT5/GR transcriptional synergy is that STAT5 recruits GR to the promoter and allows the strong transactivation domain of GR to supplement the weaker transactivation domain of STAT5. We initiated experiments to better understand the mechanism of transcriptional synergy between STAT5 and GR. Using electrophoretic mobility shift assays (EMSAs), immunoprecipitation, and Western blotting at various times after PRL treatment, it was discovered that GR both enhanced and prolonged the DNA-binding activity of STAT5. This was correlated with increased STAT5 tyrosine phosphorylation suggesting, therefore, that GR enhances STAT5 DNA binding by modulating the phosphorylation state of STAT5. That this enhancement may be specific to GR was demonstrated in experiments where the estrogen receptor (ER) exerted the opposite effect. Enhancement of STAT5 activity by GR is, therefore, one component of the transcriptional synergy exhibited by STAT5 and GR at the b-casein promoter. A similar mechanism may be operable at other STAT5-dependent promoters.

RESULTS Nuclear Translocation of STAT5 and GR Because nuclear translocation is essential for the actions of both STAT5 and GR, we initially analyzed whether STAT5 and GR could affect each other’s subcellular localization. COS-1 cells were transiently transfected with GR, PRL-R, and STAT5a and treated with either PRL or HC for 30 min. The subcellular localization of both proteins was examined by indirect immunofluorescence (IIF). Using an affinity-purified polyclonal antibody to STAT5a detected by a Texas Red-conjugated antirabbit secondary antibody and a monoclonal antibody to GR detected by a fluorescein isothiocyanate (FITC)-conjugated antimouse secondary antibody, we were able to identify cotransfected cells and examine the behavior of each protein. In the absence of either hormone, STAT5a (Fig. 1A) and GR (Fig. 1B) are both predominately localized in the cytoplasm. PRL treatment results in the nuclear localization of STAT5a (Fig. 1C), and HC treatment translocates GR to the nucleus (Fig. 1F) as expected. In cotransfected cells treated with only PRL, a subset of the GR in the cell is present in the nucleus in the absence of any HC stimulation (Fig. 1D). GR does not translocate to the nucleus after PRL treatment in the absence of STAT5 cotransfection (data not shown). The converse was also observed. In cells treated with only HC, a portion of the STAT5 is localized in the nucleus (Fig. 1E), and no translocation of STAT5 is seen in response to HC in the absence of GR (data not

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Fig. 1. Nuclear Translocation of STAT5 and GR COS-1 cells were transiently transfected with PRL-R, STAT5a, and GR. PRL or HC treatment (both at 1 mg/ml) was performed for 30 min. IIF was performed using a polyclonal STAT5a antibody detected by Texas Red-conjugated antirabbit secondary antibody (panels A, C, and E) and a monoclonal GR antibody (BuGR2) detected by FITC-conjugated antimouse secondary antibody (panels B, D, and F).

shown). Thus, PRL-activated STAT5 can interact with and translocate GR into the nucleus, and conversely ligand-bound GR can interact with and translocate inactivated STAT5 into the nucleus. The ability of STAT5 and GR to translocate in the nucleus after only one stimulus indicates an interaction between the two proteins. Colocalization of some of the nuclear STAT5 and GR was demonstrated by double IIF using deconvolution confocal microscopy (data not shown). GR Increases the DNA-Binding Activity of STAT5 Given the protein-protein interaction and transcriptional synergy exhibited by STAT5 and GR, we wished to determine whether there were any effects of GR on STAT5 DNA binding. To accomplish this, COS-1 cells were transiently transfected with PRL-R and STAT5a

alone or PRL-R, STAT5a, and GR. HC and PRL treatments were performed overnight and for 30 min, respectively. EMSA was performed on whole-cell extracts (WCEs) using an oligonucleotide containing the GAS site in the b-casein proximal promoter. This oligonucleotide also contains two half-palindromic GREs (1/2 GREs) mapped by in vitro DNA footprinting (33), which flank the GAS site. When STAT5a and GR were cotransfected and treatment with both hormones was performed, an increase in the intensity of the PRLinduced DNA-bound complex was observed as compared with the intensity of the same complex when only PRL-R and STAT5a were cotransfected (compare Fig. 2A, lanes 2 and 6). This complex can be almost completely supershifted with antibody to STAT5a (Fig. 2C, lane 4). The GR-dependent increase in STAT5a DNA binding was not due to an increase in STAT5a


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protein (Fig. 2B). Cotransfection of GR without HC treatment resulted in an intermediate level of STAT5 DNA binding (Fig. 2A, lane 4). GR and HC had no detectable effect on the DNA binding of STAT5a in the absence of PRL (Fig. 2A, lanes 3 and 5). GR enhanced the DNA-binding activity of STAT5b in a similar fashion to that of STAT5a (data not shown). In mammary epithelial cells, pretreatment with glucocorticoids is needed for induction of b-casein gene expression (34). For this reason, HC treatment was initially performed overnight, although this treatment regimen could facilitate indirect effects of GR. Because GR enhanced STAT5a DNA binding in the absence of HC (Fig. 2A, lane 4) and in light of data from other laboratories concerning STAT5-GR protein-protein interactions (2, 30), it is likely that GR enhancement of STAT5 DNA binding represents a direct effect of GR. Therefore, STAT5a DNA binding was examined after concurrent treatment with PRL and HC. A similar increase in STAT5a DNA binding was seen when HC and PRL were added concurrently for 30 min as compared with pretreatment overnight with HC followed by 30 min of PRL treatment (Fig. 2C, compare lanes 2 and 3). In COS-1 cells, the time of HC treatment did not influence the increase in STAT5a DNA binding imparted by GR. This is probably due to overexpression of STAT5 and GR in these cells. In other cell lines, pretreatment with HC was not sufficient to see an increase in STAT5a DNA binding (see below). These results suggest that enhancement of STAT5 DNA binding is a direct rather than an indirect effect of GR. The GR-dependent increase in STAT5a DNA binding was also observed when an oligonucleotide with both 1/2 GREs mutated was used as the probe (Fig. 2D). Thus, despite the presence of 1/2 GREs in the oligonucleotide used for EMSA, the principal GR-enhanced STAT5a complex detected in these experiments most likely does not contain stably associated GR (see Discussion). Because COS-1 cells are transformed with SV40 large T antigen and amplify plasmids containing an SV40 origin of replication, we wanted to ensure that GR enhancement of STAT5 DNA binding was not due only to overexpression. Accordingly, CHOk1 cells were transiently transfected with PRL-R, STAT5a, and GR, and PRL and HC treatment was performed as

Fig. 2. GR Enhances the DNA Binding Activity of STAT5a COS-1 cells were transiently transfected with PRL-R and STAT5a. GR was cotransfected as indicated. HC treatment (1 mg/ml) was performed overnight, and PRL treatment (1 mg/ ml) was performed for 30 min as indicated. A, WCE were used for EMSA on a 40-bp oligonucleotide containing the GAS site and surrounding sequences in the b-casein proximal promoter. The solid arrow indicates the PRL-inducible complex that contains STAT5a. The open arrow indicates free probe.

The autoradiogram shown was exposed for 16 h. The average increase in STAT 5a DNA binding imparted by GR in nine independent experiments was 73% 6 22% SEM B, Western blot of WCE using anti-STAT5a antibody. C, WCEs were used for EMSA. HC treatment was performed overnight or for 30 min as indicated. The solid arrow indicates the PRL-inducible complex that contains STAT5a. The open arrow indicates the complex supershifted with antibody to STAT5a. The autoradiogram shown was exposed for 18 h. D, WCEs were used for EMSA on an oligonucleotide containing the b-casein GAS site with flanking 1/2 GREs mutated. GR 1 HC led to a 90% increase in STAT5a DNA binding in this experiment. The autoradiogram shown was exposed for 4 h.

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indicated. Treatment with PRL 1 HC for 1.5 h gave a higher level of STAT5a DNA binding than treatment with PRL alone (Fig. 3, lane B vs. lane C). These data confirm that GR enhancement of STAT5 DNA binding is observed in transiently transfected cells in the absence of overexpression. In CHOk1 cells, GR enhancement of STAT5a DNA binding was seen only with concurrent PRL and HC treatment and was not observed with overnight HC treatment before PRL treatment (Fig. 3, lanes C vs. D). This is in contrast to the situation in COS cells and supports the theory that enhancement of STAT5 DNA binding by GR is a direct effect of the STAT5/GR protein-protein interaction.

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tide as a competitor ranging from 0.5 to a 16-fold molar excess. At a low molar excess of competitor, the GR-enhanced STAT5a complex was more resistant to oligonucleotide competition than the complex formed when only STAT5a was present (Fig. 4A, lanes 2–5 and 9–12). This was particularly evident when the unlabeled oligonucleotide was present at a 2-fold molar excess or less (Fig. 4A, lanes 2, 3, and 4 vs. lanes 9, 10, and 11). These differences are summarized quantitatively in Fig. 4B. The ability of the GR- enhanced complex to more effectively resist competition by an unlabeled oligonucleotide suggests that the GRSTAT5a interaction results in either a higher DNA af-

GR Enhancement Makes the STAT5a Complex More Resistant to Oligonucleotide Competition After demonstrating that GR enhanced STAT5 DNAbinding activity without changing the levels of STAT5 protein, we wished to determine the mechanism by which GR elicited this effect. To examine the nature of the STAT5-DNA interaction, EMSAs were performed using an increasing amount of unlabeled oligonucleo-

Fig. 3. GR Enhancement of STAT5a DNA Binding in CHOk1 Cells CHOk1 cells were transiently transfected with PRL-R, STAT5a, and GR. HC and PRL treatment (both 1 mg/ml) were performed concurrently for 1.5 h, or HC pretreatment for 24 h was performed followed by 1.5 h PRL treatment, as indicated. WCEs were used for EMSA. The STAT5a containing complex is indicated with a solid arrow. A non-PRL-inducible band used for normalization when quantitating band intensity is indicated by the open arrow. The solid arrowhead at the bottom of the gel indicates free probe. The average increase in STAT5a DNA binding imparted by GR in three independent experiments was 40% 6 10% SEM.

Fig. 4. GR Enhancement Makes the STAT5a Complex More Resistant to Oligonucleotide Competition A, EMSA using WCE from transiently transfected COS cells. The predominant STAT5a-containing complex is indicated by the solid arrow. A second STAT5a-containing complex is indicated by the open arrow. This complex may contain tetrameric STAT5a. No oligonucleotide competitor was added to lanes 1 and 8. Compared with the probe, unlabeled oligonucleotide competitor was added as follows: lanes 2 and 9, 0.53; lanes 3 and 10, 13; lanes 4 and 11, 23; lanes 5 and 12, 43; lanes 6 and 13, 83; lanes 7 and 14, 163. B, Quantitation of the complexes seen in panel A. Relative DNA binding was defined as the intensity of the predominant STAT5a-containing complex with competitor present compared with the complex intensity without any competitor and expressed as a percentage. The results shown are representative of those observed in three independent experiments.


finity or a slower dissociation rate of STAT5 from the DNA or both. GR Prolongs STAT5 Activation after PRL Treatment Next, we examined whether GR changes the rate of STAT5 inactivation and dissociation from the DNA. COS-1 cells were transiently transfected with PRL-R and STAT5a or PRL-R, STAT5a, and GR and treated with either PRL or PRL 1 HC for various lengths of time, and DNA binding was again examined by EMSA. Cotransfection with GR and treatment with both hormones resulted in increased STAT5a DNA binding for a longer period of time after PRL treatment (Fig. 5A). In the experiment shown in Fig. 5A, the GR-induced in-

Fig. 5. GR Prolongs STAT5 DNA Binding after PRL Treatment A, COS-1 cells were transiently transfected with PRL-R and STAT5a or PRL-R, STAT5, and GR. Cells were treated with PRL or PRL 1 HC for the time points indicated. The predominant STAT5a-containing complex is indicated by the solid arrow. The slower mobility STAT5a-containing complex is indicated by the open arrow. B, Quantitation of time course EMSA experiments. Each sample was normalized to a nonPRL-inducible band in the same lane. Relative DNA binding was defined as the intensity at a particular time point compared with the intensity after 30 min of the same treatment and expressed as a percentage. The 6-h time points are the averages of three independent experiments. All other time points are averages of five to six independent experiments. Error bars depict the SEM For all time points, the difference between STAT5a 1 PRL and STAT5a 1 GR 1 PRL 1 HC is statistically significant (P , 0.05).

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crease in STAT5a DNA binding after 30 min of PRL treatment was less than average: only 20% increase (Fig. 5A, lane 3 compared with lane 4). However, the differences between STAT5 alone and STAT5 and GR in combination were more pronounced at all the other time points in the experiment (Fig. 5A, lanes 5–12). Quantitation from several independent experiments is shown in Fig. 5B. The differences at 1.5 h and 3.0 h after PRL treatment were particularly striking. After 1.5 h of PRL treatment, the level of STAT5 DNA binding was approximately 60% of that seen after 30 min of PRL treatment. When GR was cotransfected and treatment with both HC and PRL was performed, 94% of the STAT5a DNA-binding activity detected after 30 min of hormonal treatment was still detected at 1.5 h. After 3 h of PRL treatment, the DNA-binding activity in cells transfected with STAT5a alone decreased to approximately 50% of the level seen after 30 min of PRL treatment. Cells cotransfected with GR and treated with both hormones still retained 80% of their original DNA-binding activity. The differences seen between the two treatment groups were statistically significant for all four time points tested (P , 0.05). STAT5 DNA binding is dependent on STAT5 dimerization. STAT5 dimerization is mediated by tyrosine phosphorylation on a critical tyrosine residue (Y700 in rat STAT5a) that participates in a phosphotyrosine/ SH2 domain interaction (10). We examined whether the differences in STAT5a DNA binding after PRL treatment imparted by GR were due to differences in tyrosine phosphorylation. WCE from COS-1 cells, transiently transfected and hormonally treated as above, were immunoprecipitated with anti-STAT5 Nterminal antibody. The immunoprecipitated material was divided equally, separated by SDS-PAGE, and examined by Western blotting. Western blots were probed in parallel with an antiphosphotyrosine antibody (Fig. 6, A and B) and anti-STAT5a C-terminal antibody (Fig. 6, C and D). When STAT5a alone was transfected, the level of STAT5a tyrosine phosphorylation was maximal after 30 min of PRL treatment and decreased rapidly thereafter (Fig. 6A). A faint complex could still be detected after 1.5 h of PRL treatment in longer film exposures (Fig. 6A, lane 4; data not shown). No tyrosine phosphorylation of STAT5 could be detected 3 h after PRL treatment. The slight discrepancy between detectable tyrosine phosphorylation and detectable STAT5 DNA binding is explained by the fact that EMSA is a much more sensitive assay than Western blotting of immunoprecipitated proteins. When STAT5a and GR were cotransfected and treatment with both PRL and HC was performed, STAT5a remained tyrosine phosphorylated for a prolonged time period after PRL treatment (Fig. 6B). The levels of total STAT5a protein did not change significantly over the course of the experiment (Fig. 6, C and D). The same control sample (labeled C) was included on all Western blots. Although the signal intensity in Fig. 6C is less than the signal intensity in Fig. 6D, the intensity of this positive control (Fig. 6, panels C and D, lanes 8 and 16)

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Fig. 6. GR Prolongs STAT5 Phosphorylation after PRL Treatment COS-1 cells were transiently transfected and hormone treated as in Fig. 5. In the experiment shown, HC treatment was performed overnight. As previously noted, this gave the same results as concurrent PRL and HC treatment in COS-1 cells. WCEs were immunoprecipitated using the STAT5 N-terminal antibody. Immunoprecipitated material was split into two portions and separated by SDS-PAGE. Panels A and B show Western blots probed with a monoclonal anti phosphotyrosine antibody. Panels C and D show Western blots of the same samples probed with anti-STAT5a C-terminal antibody. The heavy band at the bottom of panels C and D is immunoglobulin heavy chain, which appears because polyclonal antibodies were used for both immunoprecipitation and Western blotting. Extracts from COS-1 cells transiently transfected with PRL-R and STAT5a and treated with PRL, which were known to have a detectable level of tyrosine-phosphorylated STAT5a, were included as positive controls. Lanes 8 (panels A and C) and 16 (panels B and D) contain these samples.

is also decreased. The apparent difference is, therefore, caused by variation in the chemiluminence detection system and not by a difference in STAT5a protein levels. These results indicate that the proteinprotein interaction between STAT5 and GR prolongs STAT5 tyrosine phosphorylation and DNA binding after PRL treatment. This enhancement of STAT5 activity by GR is probably a contributing factor in the transcriptional synergy exhibited by STAT5 and GR. GR Enhances the DNA-Binding Activity of STAT5a2 STAT5a2 is a naturally occurring alternative splice form of STAT5a. STAT5a2 exhibits the same properties and is only a few amino acids shorter than STAT5aD750, an artificially generated construct, which has been extensively studied (29) (S. L. Wyszomierski and J. M. Rosen, unpublished results). The transactivation domain of STAT5a is missing in both of these proteins, and this region has been postulated to be important for interaction with a tyrosine phosphatase. When GR is cotransfected, carboxytruncated STAT5 is converted from a dominant negative factor into a positive transactivator. Therefore, the carboxy-terminal region of STAT5 is not required for transcriptional synergy with GR (31, 32). We were interested in examining whether GR could also influence the DNA-binding activity of STAT5a2. COS-1 cells

were transiently transfected with PRL-R and STAT5a2, or PRL-R, STAT5a2, and GR, and cells were treated with HC and PRL as indicated. Unlike STAT5a, STAT5a2 exhibits a basal level of DNA binding in the absence of PRL (Fig. 7, lane A). This low level of STAT5a2 DNA binding in the absence of PRL was dramatically enhanced almost 10-fold when GR was cotransfected (Fig. 7, lane C) and was further enhanced by 100% when treatment with HC was performed (Fig. 7, lane E). STAT5a2 exhibited a very high level of DNA binding after 30 min PRL treatment (Fig. 7, lane B). The level of STAT5a2 binding after PRL treatment was increased when GR was cotransfected to twice the level seen with STAT5a2 alone (Fig. 7, lane D). Treatment with both HC and PRL did not lead to an additional enhancement of STAT5a2 binding (Fig. 7, lane F). This may be because the DNA-binding activity was already maximal and could not be further increased. Carboxy-truncated STAT5b behaved in a similar manner (data not shown). Therefore, carboxyterminal sequences of STAT5 that are not required for GR-STAT5 transcriptional synergy are also not required for GR enhancement of STAT5 DNA binding. ER Exhibits Different Effects than GR on STAT5 Phosphorylation and DNA Binding We next wished to examine whether enhancement of STAT5 phosphorylation and DNA binding was specific


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Fig. 7. GR Enhances the Binding Activity of STAT5a2 in the Absence of PRL Treatment COS-1 cells were transiently transfected with PRL-R and STAT5a2 with and without GR as indicated. HC treatment was performed overnight, and PRL treatment was performed for 30 min as indicated.

to GR or if it could be conferred by another steroid hormone receptor. We used the estrogen receptor (ER) for this purpose. COS-1 cells were transiently transfected with STAT5a, PRL-R, and ER and treatment with PRL and E2 was performed as indicated. At all time points examined, cotransfection with ER resulted in decreased STAT5a DNA binding activity compared with STAT5a alone (Fig. 8A, compare lanes 4 and 6 with lanes 3 and 5). STAT5a DNA binding was decreased by ER even in the absence of E2 (Fig. 8A, lane 7) similar to the ligand-independent stimulatory effect of GR. ER did not affect STAT5 DNA binding in the absence of PRL (Fig. 8A, lanes 2 and 8). An antibody that recognizes only STAT5, which is tyrosine phosphorylated on tyrosine 700, was used to examine STAT5a tyrosine phosphorylation using direct Western blotting (A. V. Kazansky, E. B. Kabotyanski, J. Yeh, S. L. Wyszomierski, and J. M. Rosen, submitted). The decrease in STAT5a DNA binding caused by ER was reflected by a parallel decrease in STAT5a tyrosine phosphorylation (Fig. 8B; compare lanes 4 and 6 to lanes 3 and 5). ER did not affect the level of STAT5a protein (Fig. 8C). Finally, the effect of ER on STAT5 DNA binding was examined at different times after PRL treatment to determine whether ER affected the rate of STAT5 inactivation. STAT5a DNA binding after PRL stimulation decreased with similar kinetics in cells cotransfected with STAT5a and ER and cells transfected with STAT5a alone (Fig. 8D). Therefore, the GR-dependent enhancement of STAT5 tyrosine phosphorylation is clearly not a general effect of all steroid hormone receptors. GR enhanced STAT5 phosphorylation and DNA binding by decreasing the rate of STAT5 dephosphorylation, whereas ER decreased the initial level of STAT5 tyrosine phosphorylation but had no detectable effect on the kinetics of dephosphorylation.

DISCUSSION These experiments have demonstrated that the GRSTAT5 interaction protects STAT5 from inactivation by dephosphorylation. Accordingly, STAT5 stays bound

Fig. 8. ER Decreases the DNA-Binding Activity and Tyrosine Phosphorylation of STAT5a COS-1 cells were transiently transfected with PRL-R and STAT5a with or without ER as indicated. Cells were treated with PRL (1 mg/ml) and E2 (1 3 1026 M) as indicated. A, EMSA. B, WCE (20 mg) was separated by SDS-PAGE. Western blotting was performed using anti-STAT5Y700P antibody. C, WCEs (10 mg) were separated by SDS-PAGE, and Western blotting was performed using anti-STAT5a C-terminal antibody to verify similar protein levels between the samples. D, EMSA of STAT5a DNA binding after various treatments. Regardless of the absolute intensity, STAT5 DNA binding after 30 min was normalized to 100% for each treatment, and relative DNA binding for each time point was calculated as a percentage of the 30 min value. For STAT5a 1 ER samples, points are the averages of three experiments. For STAT5a alone and STAT5a 1 GR samples, points are the averages of five to six experiments as indicated in Fig. 5B. Error bars depict SEM. STAT5a 1 ER samples do not differ significantly from STAT5a samples for all time points. STAT5a 1 GR samples differ from the other two treatment groups in a statistically significant manner (P , 0.05) for all time points.

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to DNA for an extended period of time after PRL treatment, which may facilitate increased transcriptional activation. STAT5 and GR are clearly capable of transcriptional synergy at the b-casein promoter (2, 32). This transcriptional synergy is the result of a pleiotropic mechanism of which GR enhancement of STAT5 DNA binding is only one component. STAT5 and GR interaction and nuclear translocation as a consequence of a single ligand-induced activation of a respective signaling pathway has now been demonstrated both by IIF in this study and quantitative Western blots using nuclear and cytoplasmic fractions from HC11 cells (30). In the latter study, these investigators found 1.5 times more GR in the nucleus of HC11 cells after PRL treatment compared with no hormone treatment. A similar increase in nuclear STAT5 was observed when only glucocorticoid treatment was performed. Thus, it is conceivable that, under some circumstances, inactive STAT5 and GR may be in the nucleus complexed with their activated partner. The physiological significance of this observation remains to be determined. Protein tyrosine phosphatases located in the nucleus have been implicated in STAT inactivation (36). Our data suggest that when STAT5 is complexed with GR, protein-protein interactions decrease the affinity of STAT5 for a deactivating phosphatase. This could be through a mechanism involving steric hindrance, particularly if GR and the phosphatase interact with the same region or nearby regions of STAT5. Because GR can enhance the DNA binding activity of STAT5a2, it appears that the carboxy terminus of the STAT5 is not necessary for this effect. Although the carboxy terminus of STAT5 has been suggested as a potential STAT5-phosphatase interaction site (29), the aminoterminal sequences of STAT proteins may also be important for phosphatase interactions (37). The amino terminus is a region of high sequence homology between the STAT proteins. Eight a-helical regions are predicted, based on amino acid sequence, in the amino termini of all STATs. The crystal structure of STAT4 revealed that these helices form a hookshaped structure that potentially can mediate a variety of protein-protein interactions (38). When STAT1 is truncated by 61 amino acids at the amino terminus, tyrosine dephosphorylation is inhibited. This results in a high basal level of phosphorylation and prolonged activation after stimulation. Arg 31 and Glu 39 have been shown to be particularly important and are conserved in all STAT proteins including STAT5a and STAT5b (37). In addition, a region slightly upstream of the STAT5 DNA-binding domain (DBD) has been implicated in STAT5 dephosphorylation. The double mutation of amino acid 299 from histidine to arginine and amino acid 711 from serine to phenylalanine resulted in prolonged tyrosine phosphorylation of STAT5 after cytokine stimulation (39). Once the regions of STAT5 that interact with GR are more precisely delineated and the specific nuclear tyrosine phosphatase is de-

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fined, it should be feasible to test directly this hypothesis. The data presented in this study are consistent with the hypothesis that GR decreases the affinity of STAT5 for an inactivating phosphatase, which then leads to enhancement and prolongation of STAT5 DNA binding. Alternatively, GR interaction could increase the affinity of STAT5 for DNA, thereby making it less accessible to an inactivating phosphatase resulting in prolonged tyrosine phosphorylation. The latter hypothesis appears less likely, because these experiments are performed in cells transfected with STAT5a, GR, and PRL-R but not a target promoter sequence. Both COS and CHO cells would be required to have an accessible endogenous promoter containing a STAT5-binding site, and possibly 1/2 GREs similar to those found in the b-casein promoter for the second hypothesis to explain the effect. Although protein-protein interactions between STAT5 and GR are well established, the possibilities either that GR is prolonging STAT5 tyrosine phosphorylation by interacting directly with a phosphatase or perhaps acting upstream to increase STAT5 activation via the PRL-R and JAK2 cannot yet be eliminated. Cotransfection with ER led to a decrease in tyrosine phosphorylation and DNA binding of STAT5 without affecting STAT5 protein levels. The physiological significance and mechanism of ER modulation of STAT 5 are not understood at present, but may be relevant for a number of genes that are regulated by both cytokines and estrogens. The observed decrease in STAT5 tyrosine phosphorylation is probably the result of a protein-protein interaction involving ER because estradiol was not required to observe this effect. A direct or indirect interaction between the ER and STAT5 would be the most plausible explanation, but this remains to be established. ER clearly does not affect the rate of STAT5 dephosphorylation. Therefore, the decrease in phosphorylation and DNA binding is probably the result of an inhibition of STAT5 activation. Thus, ER may decrease the interaction between STAT5 and PRL-R or the interaction between STAT5 and JAK2. Alternatively, if STAT5 and ER interact in a ligandindependent fashion, similar to STAT5 and GR (30), the decrease in STAT5 tyrosine phosphorylation could result from ER sequestration of a subpopulation of STAT5 in the nucleus away from receptor/JAK2 activation at the cell membrane. Although we do not understand how ER affects STAT5, the experiments presented here demonstrate that steroid receptors other than GR impinge on the JAK/STAT signaling pathway. Progesterone is known to inhibit b-casein expression during pregnancy (40). PR has been found to inhibit STAT5 induction of b-casein in transiently transfected CHOk1 cells, and there is evidence of an interaction between STAT5 and PR (E. K. Gass and D. P. Edwards, personal communication). Androgen receptor may also enhance STAT5 tyrosine phosphorylation in a manner similar to GR (S. L. Wyszomierski and J. Rosen, unpublished observations).


The same steroid receptor may also exert selective effects on different STAT proteins. For example, it has been reported that GR interacts and transcriptionally synergizes with both STAT5 (2) and STAT3 (41) on STAT-responsive promoters. An interesting difference occurs on the GR-responsive mouse mammary tumor virus (MMTV) promoter, however. Here STAT3 acts a coactivator with GR to enhance transcription at the MMTV promoter (41), while STAT5 inhibits the MMTV promoter, presumably by sequestering GR in a complex that is incapable of transactivation at the MMTV promoter (2). Further analysis is likely to reveal a plethora of steroid receptor-STAT interactions, each with specific effects and implications based on the proteins involved, the cell type, and the promoter. While glucocorticoids are known to be essential lactogenic hormones, the mechanisms by which they regulate milk protein gene expression have not been completely defined. Both direct and indirect mechanisms appear to be responsible for steroid hormone regulation of b-casein gene expression. PRL and HC have been demonstrated to act by kinetically distinct mechanisms in mammary epithelial cells (34). Pretreatment with glucocorticoids is essential for PRL induction of b-casein gene transcription. This effect is gradually increased with longer glucocorticoid pretreatments, is rapidly reversed when glucocorticoids are withdrawn, and requires ongoing protein synthesis (34, 42). Glucocorticoids may also modulate b-casein gene transcription through alterations in the levels of different C/EBPb (CCAAT-enhancer binding protein b) isoforms in an indirect manner requiring new protein synthesis (43). In contrast to this indirect effect, the rapid transcriptional synergy seen by STAT5 and GR is a direct effect on b-casein gene transcription. The GR-enhanced EMSA complex detected in these experiments is not likely to contain stably associated GR, despite the presence of 1/2 GREs in the oligonucleotide used for EMSA. A slower mobility EMSA complex was not detected after expression of GR and STAT5 as compared with STAT 5 alone; antibodies to GR had no effect on the mobility of the EMSA complex; and a consensus GRE did not compete in oligonucleotide competition experiments (data not shown). This is consistent with results published by Cella et. al. (30), who were also unable to detect STAT5 and GR in a stable DNA-bound complex on the b-casein promoter by EMSA. However, by incubating extracts with a b-casein oligonucleotide followed by immunoprecipitation with antibody to GR, both GR and STAT5 have been detected in a DNA-bound complex on the b-casein promoter. The formation of this complex was dependent on the presence of an intact GAS site (30). It is still somewhat controversial whether or not GR binding to the b-casein promoter is necessary for the observed transcriptional synergy between STAT5 and GR. There are no palindromic GREs in the b-casein promoter. However, several 1/2 GREs have been mapped in the promoter by in vitro DNAseI footprinting

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with purified GR (33). The GR DBD alone is capable of binding to some of these 1/2 GREs as a monomer. Mutation of several of the 1/2 GREs individually and in combination abolishes STAT5/GR transcriptional synergy in COS cells. Mutation of the three 1/2 GREs found between 2180 and 261 of the b-casein promoter strongly reduced the synergistic effects of PRL and HC seen in HC11 cells. However, when GR mutants containing either mutated DBDs or the DBD of ER were cotransfected with STAT5 into COS-7 cells, these mutated receptors were still capable of transcriptional synergy with STAT5 (31). The level of synergy was, however, decreased compared with wildtype GR. These results suggest that GR binding to the 1/2 GREs in the b-casein promoter enhances, but is not absolutely required for, STAT5/GR transcriptional synergy. Of interest in this regard is the recent observation that only a subset of GR functions in vivo were affected by the lack of GR DNA binding in mice where wild-type GR was replaced with DNA binding-defective GR by gene targeting (44). It will be of interest to analyze mammary gland development and functional differentiation in these mutant mice. While the GR-dependent enhancement of STAT5 phosphorylation and DNA binding is most likely a result of protein-protein interaction between STAT5 and GR, and does not require binding to the b-casein promoter, binding of both proteins to the b-casein promoter may amplify in vivo the protective effect of GR on STAT5 phosphorylation seen in these in vitro experiments. Because DNA binding is not needed for GR to enhance STAT5 phosphorylation, this proteinprotein interaction has the potential to effect the transcription of any gene induced by STAT5. It could have a greater impact, however, on promoters that are capable of binding both STAT5 and GR, such as the milk protein and acute phase gene promoters. In the b-casein gene STAT5, GR, and C/EBPb all participate in transcriptional activation as part of a composite response element (CoRE) (11, 12, 32, 43, 45, 46). In the presence of lactogenic hormones, transcriptional synergy is conferred by a pleiotropic mechanism involving cooperation of the transactivation domains of STAT5 and GR (2, 31) and enhancement of STAT5 DNA binding by GR. GR has also been shown to interact directly with C/EBPb (47), so protein-protein interactions are likely to stabilize the binding of each individual transcription factor to its response element, thereby creating a stable activation complex. STAT5, GR, and C/EBPb have all been shown to interact with p300/CBP (48–50). Recruitment of coactivators and cointegrators like p300 or CBP to the promoter is likely to be a critical component of transcriptional synergy. Interactions between different classes of transcription factors at CoREs result in highly specific regulation of gene expression (Ref. 51) and references therein), as exemplified by the transcriptional synergy exhibited by GR and STAT5 at the b-casein CoRE. The observation that GR enhances STAT5 activation by prolonging its tyrosine phosphor-

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ylation is an interesting example of how transcription factors in CoREs may be able to modulate each other’s activities.

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was used instead of regular DMEM throughout these experiments. IIF

Rat STAT5a and rat STAT5b cDNAs were subcloned into the pRcCMV expression vector (Invitrogen, Carlsbad, CA) and kindly provided by Guoyang Luo and Dr. Li-yuan Yu-Lee (Baylor College of Medicine, Houston, TX). The STAT5a2 expression construct was generated through subcloning by placing N-terminal sequences of STAT5a with C-terminal sequences of STAT5a2 in the pcDNA3 expression vector. This was done because only the C-terminal sequences of STAT5a2 were isolated from the original screening of a cDNA library prepared from RNA isolated from the rat mammary gland at day 2 of lactation (15). A unique SalI restriction site was used. Subsequently, 1763 nucleotides, which encoded exclusively 39-untranslated sequences for STAT5a2, were removed from the construct to eliminate problems due to splicing of the cDNA construct. A rat GR expression plasmid (VARO) and a monoclonal antibody to GR (BuGR2) were kindly provided by Dr. Donald Defranco (University of Pittsburgh, Pittsburgh, PA). A human ER expression plasmid (pCR3.1 hER) was kindly provided by Dr. Zafar Nawaz (Baylor College of Medicine). This plasmid was generated by placing the SalI fragment of pRST7hER, which contains the cDNA for ER (52) into the pCR3.1 vector (Invitrogen). All plasmids were purified using a Qiagen DNA maxi-prep kit (Qiagen, Valencia, CA).

Cells were cultured and transfected on glass coverslips, coated with poly-D-lysine (1 mg/ml, mol wt 70,000–150,000, Sigma). The coverslips were placed on ice, rinsed two times with ice-cold PBS, and then fixed with 4% paraformaldehyde in PEM buffer (80 mM 1,4-piperazinediethane sulfonic acid, 1 mM EGTA, 1 mM MgCl2, pH 6.9) for 30 min. After fixing, all incubations and washes were performed at room temperature. The coverslips were washed three times with PEM and incubated with PEM 1 1 mg/ml NaBH4 two times for 5 min followed by washing three times with PEM. Cells were permeablized with 0.5% Triton X-100 in PEM for 10 min. The coverslips were washed three times with PEM and once with TBST (100 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween-20). Before immunostaining, the coverslips were blocked for 1 h with TBST 1 5% nonfat dry milk (NFDM) (Carnation, Glendale, CA). The coverslips were incubated with both primary antibodies diluted in TBST 1 5% NFDM for 1 h. Anti-STAT5a C-terminal antibody was used at a 1:200 dilution. BuGR2, a monoclonal antibody to GR, was used at a 1:500 dilution. After washing five times with TBST, coverslips were incubated for 30 min in the dark with both secondary antibodies, antirabbit IgG conjugated with Texas Red (Southern Biotechnology Associates, Inc., Birmingham, AL), and antimouse IgG conjugated with FITC (Pierce, Rockford, IL). Both secondary antibodies were diluted 1:1,000 in TBST 1 5% NFDM. The coverslips were then washed five times with TBST and mounted using Vectashield mounting media containing 49,6diamidino-2-phenylindole hydrochloride (Vector, Burlingame, CA). Images were obtained by fluorescent microscopy (Zeiss Axiophot; Carl Zeiss, Thornwood, NY).

Cell Culture and Transfections

Preparation of WCEs

DMEM, trypsin-EDTA, donor horse serum, and glutamine were purchased from JRH Biosciences (Lenexa, KY). DMEM/ F12 and phenol red-free DMEM were purchased from GIBCO-BRL (Gaithersburg, MD). FBS was purchased from JRH Biosciences and Summit Biotechnologies (Fort Collins, CO). Gentamicin, insulin, apo-transferrin, HC, and b-estradiol were purchased from Sigma (St. Louis, MO). Ovine PRL (lot AFP-10677C) was kindly provided by the National Hormone and Pituitary Program (Bethesda, MD). COS-1 cells and CHOk1 cells were obtained from the ATCC (Manassas, VA). COS-1 cells were routinely passaged in DMEM 1 10% FBS in the presence of gentamicin. COS-1 cell transfections were performed 1 day after passaging the cells using a lipofectamine (GIBCO-BRL) protocol. DNA (10–15 mg) and 20 ml of lipofectamine were used per 100-mm plate. Transfections were performed according to the manufacturer’s instructions. After transfection, cells were maintained in DMEM 1 10% charcoal-stripped horse serum with gentamicin and pretreated with 5 mg/ml insulin for 24–48 h. The charcoal treatment was performed to remove endogenous steroids from the serum. Treatment with HC (1 mg/ml), b-estradiol (1 3 1026 M) and/or ovine PRL (1 mg/ml) was performed as indicated. CHOk1 cells were routinely passaged in McCoy’s 5a media 1 10% FBS in the presence of gentamicin. Transfection of CHOk1 cells was performed similarly to the COS cells, but serum-free DMEM/F12 supplemented with 5 mg/ml insulin and 10 mg/ml apo-transferrin was used throughout the experiment. These serum-free conditions for CHOk1 cells represent a minor modification to a previously reported serum-free media for CHOk1 cells (53). Lipofectamine (10 ml per plate) was used. For COS-1 transfections done to compare the effects of estrogen receptor to the effects of GR, Superfect reagent (Qiagen) was used for transfection according to the manufacturer’s instructions. DMEM without phenol red

Cells were washed twice with HBSS or PBS without calcium and magnesium (PBS) to remove media and serum. HBSS or PBS (750 ml) was added per 100-mm tissue culture dish. Cells were detached by scraping and transferred to an Eppendorf tube. Cells were pelleted briefly at 15,000 rpm at 4 C. Cell pellets were routinely frozen in liquid nitrogen and stored at 270 C before extract preparation. Cell pellets were resuspended in 2–3 volumes of 400 mM Wu buffer (400 mM NaCl, 10 mM HEPES, pH 7.4, 1.5 mM MgCl2, 0.1 mM EGTA, 5% glycerol, 1 mM dithiothreitol) supplemented with 2 mg/ml aprotinin, 2 mg/ml benzamidine, 2 mg/ml antipain, 2 mg/ml soybean trypsin inhibitor, 1.5 mg/ml leupeptin, 1 mM sodium orthovanadate, and 1 mM sodium molybdate by repeated pipetting. Most inhibitors were purchased from Sigma. Antipain was purchased from Boerhinger Mannheim (Indianapolis, IN). Resuspended cell pellets were incubated on ice for 10–15 min. Mixing by pipetting was repeated once during the incubation. Extracts were centrifuged at 15000 rpm at 4 C for 10 min to remove cellular debris. Protein levels were determined using Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA). All extracts were aliquoted, frozen in liquid nitrogen, and stored at 270 C. Each aliquot was thawed only once.

MATERIALS AND METHODS Plasmids

EMSA An oligonucleotide encompassing the b-casein GAS site and flanking 1/2 GREs was used for EMSA. The sequence of the coding strand was as follows: TAATCATGTGGACTTCTTGGAATTAAGGGACTTTT. The sequence for the coding strand of the oligonucleotide with mutated 1/2 GREs was as follows: TAATCAAGCTTACTTCTTGGAATTAACAGACTTTT. Oligonucleotides were designed with 4-bp overhangs and were la-


beled by filling in the overhangs with [32P]deoxynucleoside triphosphates (New England Nuclear LifeScience Products, Boston, MA). Labeled probe was separated from unincorporated nucleotides using either G-50 spin columns (Boerhinger Mannheim) or P-6 Micro Bio-Spin columns (Bio-Rad) according to the manufacturers’ instructions. WCEs were diluted 1:4 with no-salt Wu buffer supplemented with inhibitors (same as above but without NaCl) to adjust the salt concentration to 100 mM. Five to 10 mg of total protein were used per reaction. Wu buffer (100 mM) supplemented with inhibitors (same as above with 100 mM NaCl instead of 400 mM) was added so the total volume was 10 ml. Poly (dI)-poly (dC) (Pharmacia, Piscataway, NJ) was added (2 mg/reaction). Samples were incubated on ice for 30 min. When competitor oligonucleotides or antibodies were used, they were included with the extracts during this incubation. Competitors were added to specific concentrations as indicated. For all antibodies, 1 ml/reaction was used. Five microliters of binding mix [2.5 mg/ml BSA, 4% Ficoll 400, 10% glycerol, 50 mg/ml p (dN) 5 (Pharmacia)] containing approximately 100 ng labeled probe were added per reaction. Reaction samples were incubated on ice for 15 min to allow binding to occur. Reactions were resolved on 5% polyacrylamide (38:2 acrylamide-bis-acrylamide ratio) gels containing 0.253 TBE and 2.5% glycerol run at 250–275 V at 4 C. EMSAs were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Statistical significance was determined using a two-way, independent t test. Immunoprecipitation and Western Blots Protein A-trysacryl (Pierce) was prepared by washing three times with RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1% NP-40, 0.25% sodium deoxycholate) and resuspended in RIPA supplemented with inhibitors (1 mM dithiothreitol, 2 mg/ml aprotinin, 2 mg/ml benzamidine, 2 mg/ml antipain, 2 mg/ml soybean trypsin inhibitor, 1.5 mg/ml leupeptin, 1 mM sodium orthovanadate, and 1 mM sodium molybdate) to twice the original volume. Total protein (400 mg per sample) was used. The total volume was brought to 350–400 ml with RIPA supplemented with inhibitors. Extracts were precleared by incubation with 40 ml protein A-trysacryl for 30 min at 4 C with rocking. STAT5 N-terminal antibody (Santa Cruz STAT5 N-20, Santa Cruz, CA) was used at a 1:100 dilution. Antibody was incubated for 3 h with the extracts at 4 C with rocking. Protein A-trysacryl (60 ml) was then added, and the samples were incubated overnight at 4 C with rocking. The resin was washed three times with RIPA buffer supplemented with inhibitors, and bound proteins were eluted by boiling in SDS sample buffer for 10 min. Proteins were separated by standard SDS-PAGE techniques utilizing 3% stacking gels and 7.5% running gels. They were transferred to Immobilon-P polyvinylidene fluoride membranes (Millipore, Bedford, MA) overnight at 90 mA. Western blots were done using standard protocols (43) with STAT5a affinity-purified antibody (15) at a 1:5,000 dilution, STAT5Y700P affinity-purified antibody at a 1:400 dilution, or PY20 (Transduction Laboratories, Lexington, KY) at a 1:500 dilution. STAT5Y700P is an affinity-purified rabbit polyclonal antibody that is specific for STAT5 phosphorylated on tyrosine 700. It does not recognize STAT5 that is not phosphorylated on this residue (A. V. Kazansky, E. B. Kabotyanski, J. Yeh, S. L. Wyszomierski, and J. M. Rosen, submitted). For phosphotyrosine blots, modified TBST (10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween 20) 1 1% BSA was used for blocking and incubation with the primary antibody. Biotinylated goat antirabbit IgG, biotinylated goat antimouse IgG, and streptavidinhorseradish peroxidase were purchased from Calbiochem (La Jolla, CA).

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Acknowledgments We would like to thank Dr. Alexander Kazansky for technical advice and reagents and Dr. Mike Mancini for assistance with IIF microscopy. We would also like to thank Drs. Li-yuan Yu-Lee and Nancy Weigel for critical reading of the manuscript.

Received July 24, 1998. Revision received October 2, 1998. Accepted October 20, 1998. Address requests for reprints to: Jeffrey M. Rosen, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030-3498. E-mail: jrosen@bcm. tmc.edu. These studies were supported by NIH Grant CA-16303 from the NIH. S.L.W. was supported by a breast cancer training grant from the Department of Defense (DAMD 17–94-J-4204).

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