The Protein Tyrosine Phosphatase-PEST Is Implicated in the Negative Regulation of Epidermal Growth Factor on PRL Signaling in Mammary Epithelial Cells

Kay Horsch, Michael D. Schaller and Nancy E. Hynes

Friedrich Miescher Institute, CH-4002 Basel, Switzerland; and Department of Cell Biology and Anatomy, University of North Carolina (M.D.S.), Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: Friedrich Miescher Institute, R-1066.206, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. E-mail: nancy.hynes{at}fmi.ch


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Treatment of HC11 mammary epithelial cells with the lactogenic hormone PRL promotes differentiation and induction of milk protein gene expression via stimulation of the Janus kinase (JAK)/signal transducer and activator of transcription pathway. We have previously shown that autocrine activation of epidermal growth factor (EGF) receptor interferes with normal PRL-induced differentiation. Here we show that PRL activation of JAK2 was dramatically reduced in HC11 cells pretreated with EGF, demonstrating that the target of EGF receptor activation is JAK2 kinase. Using an in-gel protein tyrosine phosphatase (PTP) assay, we observed that the activity of a 125-kDa PTP was up-regulated in HC11 cells in response to EGF. A specific antiserum was used to demonstrate that the 125-kDa PTP was PTP-PEST and to show that EGF treatment of HC11 cells led to an increase in the level of PTP-PEST. In intact HC11 cells, PTP-PEST was constitutively associated with JAK2, and in response to EGF treatment there was an increased level of PTP-PEST in JAK2 complexes. An in vitro phosphatase assay, using PRL-activated JAK2 as the substrate and lysates from HC11 cells as the source of PTP-PEST, revealed that JAK2 could serve as a PTP-PEST substrate. However, in intact cells the regulation of JAK2 by PTP-PEST was complex, since transient overexpression of PTP-PEST had a negligible effect on PRL-induced JAK2 activation. EGF’s negative influence on JAK2 activity was blocked by actinomycin D treatment of HC11 cells, suggesting that EGF induced a protein that mediated the effects of PTP-PEST on JAK2. In support of this model, PTP-PEST-containing lysates from EGF-treated HC11 cells dephosphorylated JAK2 to a greater extent than lysates prepared from control cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MAMMARY GLAND differentiation requires the coordinated action of growth factors and hormones that promote morphological development and functional differentiation. Mammary cancer results from genetic changes that alter these normal processes. To probe the molecular events contributing to normal mammary gland development and cancer, in vitro cell culture systems have proven invaluable. The HC11 mouse mammary cell line is a useful model system for studying mammary cell differentiation, as treatment of the cells with the lactogenic hormones, PRL and glucocorticoids, leads to the production of several milk proteins (1, 2, 3). The intracellular targets of PRL are Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5). Ligand-induced activation of PRL receptor (PRLR) leads to stimulation of the JAK2 tyrosine kinase that phosphorylates STAT5 on a conserved tyrosine residue, leading to STAT5 dimerization, nuclear migration, and stimulation of target gene transcription, including the gene encoding the milk protein, ß-casein (4, 5, 6).

We have previously reported that hormonal induction of ß-casein expression is blocked by the addition of epidermal growth factor (EGF) to the lactogenic hormone-containing medium (7). Furthermore, in oncogene-transformed HC11 cells displaying autocrine activation of the EGF receptor (EGFR), PRL-induced differentiation is also inhibited (8, 9). Considering the importance of the EGF-related family of ligands and their receptors in mammary gland development and in human cancer (10, 11, 12, 13), we have further explored the mechanism underlying the EGF effect on differentiation. PRL-induced activation of the JAK2 kinase was dramatically reduced in cells pretreated with EGF, suggesting that EGF targets the PRLR-associated kinase. We found that a cytosolic protein tyrosine phosphatase (PTP), PTP-PEST, is up-regulated in EGF-treated HC11 cells. PTP-PEST, which is termed after proline glutamine serine threonine-rich motifs (PEST sequences), is ubiquitously expressed (14, 15, 16) and is known to have functions in focal adhesion breakdown, cell spreading, and cell motility (17, 18). PTP-PEST was able to dephosphorylate JAK2 in an in vitro phosphatase assay. In vivo, in HC11 cells we found that JAK2 and PTP-PEST are constitutively complexed, and EGF treatment of the cells leads to elevated PTP-PEST expression and an increased association with JAK2. Moreover, EGF appears to induce an as yet unknown protein, which has a permissive effect on PTP-PEST, allowing it to target and inhibit JAK2 in the PRLR complex. The results presented here show that PRL signaling in mammary cells is controlled by a novel mechanism that appears to involve PTP-PEST, a PTP that has not previously been implicated in down-regulation of cytokine signaling. Considering that many breast tumors display constitutive autocrine activation of the EGFR due to production of one or more of its ligands (11), this pathway may contribute to the differentiation block, which is a characteristic of mammary cancers.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
STAT5 DNA Binding Is Negatively Regulated by EGF
In HC11 cells, PRL binding to its receptor leads to JAK2-mediated phosphorylation of STAT5 on a conserved tyrosine residue; Tyr694 for STAT5a and Tyr699 for STAT5b (19). The activated STAT5 proteins dimerize and enter the nucleus, where they bind to a {gamma}-interferon-activated sequence-like site in the promoter of the ß-casein gene, stimulating its transcription (4, 5, 6, 20). To investigate the lactogenic hormone-induced formation of STAT5-DNA complexes in EGF-treated HC11 cells, EMSAs were performed using nuclear extracts from hormone-treated cells and the STAT5-binding site from the ß-casein promoter as a probe. The cultures were treated for 16 h with EGF before adding lactogenic hormones for 15 min, a time when STAT5 DNA binding is strong and easily measurable (20). The EMSAs were quantified, revealing that STAT5-binding activity was decreased by 54% in the EGF-treated cultures (Fig. 1AGo, lane 2 vs. lane 4). Considering the importance of phosphotyrosine for STAT5 DNA binding, we examined the possibility that EGF induced a PTP. HC11 cultures were treated with the PTPase inhibitor, orthovanadate. At a high concentration (1000 µM), orthovanadate nonspecifically induced STAT5 DNA binding in the absence of lactogenic hormones (Fig. 1CGo, lane 13). However, at 100 µM, orthovanadate had no effect on basal or lactogenic hormone-induced STAT5 DNA binding (Fig. 1BGo, lane 9). Importantly, the block in STAT5 DNA binding observed in EGF-treated cultures was essentially reversed in cultures treated with 100 µM orthovanadate. Quantification revealed that binding of STAT5 from vanadate-treated cultures reached 88% of the control level (Fig. 1A, lane 5 vs. lane 2). The results suggest that the negative effect of EGF on STAT5 may ensue through activation of a PTP, which targets the Tyr residues essential for DNA binding.



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Figure 1. EGF Inhibits the Induction of STAT5 DNA Binding Activity

A and B, Competent cultures of HC11 cells that had either been incubated 16 h with EGF (+) or left untreated (-) were treated for 15 min with lactogenic hormones (DIP; +) or left untreated (-) before lysis. In some cultures vanadate (100 µM) was added 90 min before lysis. In the experiment shown in C, competent HC11 cultures were treated with different concentrations of vanadate 90 min before lysis. Nuclear extracts were prepared, and EMSAs were performed using a 30-bp double stranded oligonucleotide encompassing the STAT5 DNA-binding site of the rat ß-casein promoter as a probe. The specific STAT5 bandshift and the free probe are shown.

 
EGF Inhibits Lactogenic Hormone-Induced JAK2 Kinase Activity
To test for effects of PTPs on the JAK/STAT pathway, we examined the phosphotyrosine content of STAT5 and the other members of this pathway, JAK2 and PRLR. Lysates were prepared from control cells treated for 15 min with lactogenic hormones [10-6 M dexamethasone, 5 µg/ml insulin, and 5 µg/ml ovine PRL (DIP)] or cells pretreated for 16 h with EGF before lactogenic hormone addition. STAT5, JAK2, and PRLR were each immunoprecipitated and probed for phosphotyrosine content. The expression level of PRLR, JAK2, and STAT5 was essentially the same in control and EGF-treated cultures (Fig. 2AGo, lower three panels). Notably, all three proteins showed a 60–80% reduction in their phosphotyrosine content in EGF-treated compared with control cultures (Fig. 2AGo, upper panels; quantified in Fig. 2BGo). The lysates were also probed directly with phospho-JAK2 antiserum, which recognizes phospho-Tyr1007 located in the regulatory loop of JAK2 (21), and with phospo-STAT5-specific serum, which recognizes the JAK2 phosphorylation sites on STAT5a/b. The phosphorylation of both proteins was decreased by more than 90% in the EGF-treated cultures (Fig. 2AGo, upper right panels; quantified in Fig. 2BGo).



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Figure 2. EGF Negatively Regulates Lactogenic Hormone Activation of the JAK/STAT Pathway

A, Competent cultures of HC11 cells that had either been incubated 16 h with EGF (+) or left untreated (-) were treated for 15 min with lactogenic hormones (DIP; +) or left untreated (-) before lysis. Whole cell lysates were used for immunoprecipitation of PRLR, JAK2, and STAT5a. Immunoprecipitates were analyzed by 7.5% SDS-PAGE, blotted onto a polyvinylidene difluoride membrane, and probed with phosphotyrosine-specific monoclonal antibody. After stripping, the membranes were reprobed with the specific antibodies to control for loading. The same lysates were directly analyzed with phospho-specific antibodies for JAK2 and STAT5. B, Quantification of the phosphorylations shown directly above in A. ImageQuant software (Molecular Dynamics, Inc.) was used to quantify the phosphorylations and to normalize it to protein. Values shown are relative to DIP-induced phosphorylation ({square}). C, JAK2 kinase activity in HC11 cultures treated as described in A. Where indicated, vanadate (100 µM) was added 90 min before lysis. Immunoprecipitates of JAK2 were incubated in kinase buffer with ATP for 30 min. After the kinase reaction was stopped, the autophosphorylated immunocomplexes were analyzed by 7.5% SDS-PAGE, blotted onto a polyvinylidene difluoride membrane, and probed with a phosphotyrosine-specific monoclonal antibody. After stripping, the membrane was reprobed with JAK2-specific antiserum. The lowest panel shows a direct Western blot with phospho-specific JAK2 antibody. D, Quantification of JAK2 kinase activity ({blacksquare}) and the corresponding phospho-JAK2 levels ({square}) shown in C. ImageQuant software was used to quantify the phosphorylations and to normalize it to protein. Values shown are relative to DIP-induced activity or phosphorylation.

 
The preceding results strongly suggest that in HC11 cells JAK2 kinase is the target for negative regulation by EGF. We performed in vitro kinase assays with JAK2 (22, 23) to directly test this possibility. JAK2 was immunoprecipitated from HC11 cultures treated with various combinations of lactogenic hormones, EGF, and orthovanadate. The immunoprecipitates were incubated with kinase buffer in the presence of cold ATP, and JAK2 activity was measured by determining its phosphotyrosine content in a Western analysis (Fig. 2CGo, upper panels). The same lysates were also probed directly with the phospho-JAK2-specific serum (Fig. 2CGo, lower panel). The activity of JAK2 isolated from cultures of lactogenic hormone (DIP)-treated cultures was increased 2-fold relative to the basal activity seen in control or EGF-treated cultures (Fig. 2CGo, lane 3 vs. lanes 1 and 2; quantified in Fig. 2DGo). Moreover, EGF treatment severely blocked lactogenic hormone induction of JAK2 activity, returning it to the basal level (Fig. 2CGo, lane 4 vs. lane 1; quantified in Fig. 2DGo). Importantly, orthovanadate had no effect on basal JAK2 activity (Fig. 2CGo, lane 6); however, it almost completely reversed (96%) the negative influence of EGF on lactogenic hormone-induced JAK2 activity (Fig. 2CGo, lane 5 vs. lane 3; quantified in Fig. 2DGo). These results strongly support the hypothesis that EGF influences the activity of a PTP that negatively regulates JAK2 activity.

A 125-kDa PTPase Has Higher Activity in EGF-Treated Cells
To test the involvement of PTPs in the EGF inhibition of JAK/STAT signaling, we employed an in-gel assay, which reveals the activity of cytoplasmic PTPs after their separation by SDS-PAGE and in-gel renaturation (24). Lysates from HC11 cells routinely displayed 10 active PTPs in the in-gel assay (not shown); the four most active PTPs migrate as proteins of approximately 50, 62, 64, and 125 kDa (Fig. 3AGo). None of these PTPs was affected by lactogenic hormone treatment. However, the activity of the 125-kDa PTP was elevated in lysates prepared from EGF-treated cells. Equal loading of the gel was confirmed by Coomassie staining (Fig. 3BGo). Immunoprecipitates of the EGFR were also examined by an in-gel PTP assay. Only one band of activity was observed, which comigrated with the activity at 125 kDa. Intriguingly, in comparison to long-term treatment (Fig. 3AGo), its activity was not affected by short-term (15 min) EGF treatment (Fig. 3CGo).



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Figure 3. A 125-kDa Protein Tyrosine Phosphatase Has Higher Activity in EGF-Treated Cells

Competent cultures of HC11 cells that had either been incubated 16 h with EGF (+) or left untreated (-) were treated for 15 min with lactogenic hormones (DIP; +) or left untreated (-) before lysis. Protein tyrosine phosphatase activities were analyzed from whole cell lysates (A) and from immunoprecipitates of EGFR (C) in an in-gel phosphatase assay, using [32P]poly-(Glu:Tyr) as the gel-embedded substrate. After SDS-PAGE, SDS was removed from the gel, proteins were renatured, and the gel was incubated in phosphatase buffer. The phosphatase reaction was terminated by Coomassie staining of the gel that also served to control for loading (B). Gels were dried, and PTP activities were visualized in A and C using a PhosphorImager (Molecular Dynamics, Inc.). C, Cells were incubated for 15 min with EGF (+). The asterisk in A marks the activities of SHP1 and SHP2.

 
The 125-kDa PTP Is PTP-PEST
Considering that PTP-PEST is approximately 125 kDa and has been reported to associate with EGFR (25), this PTP was examined more closely. PTP-PEST was specifically immunoprecipitated from lysates of HC11 cells, which had been treated with EGF and/or the lactogenic hormones. The results show that the activity of the immunoprecipitated PTP-PEST (Fig. 4AGo, upper panel) has the same electrophoretic mobility as the 125-kDa activity identified in the previous experiment (Fig. 3Go). Furthermore, PTP-PEST activity was increased 2-fold in all cultures treated with EGF, but not in cultures treated with lactogenic hormones alone (Fig. 4Go, A and B, lanes 2 and 4 vs. lane 3). There was also more PTP-PEST in the immunoprecipitates from EGF-treated compared with control HC11 cultures (Fig. 4AGo, lower panel), suggesting that the increased PTP-PEST activity seen in the EGF-treated cultures might reflect increased PTP-PEST expression.



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Figure 4. The 125-kDa Protein Tyrosine Phosphatase Is PTP-PEST

A, Competent cultures of HC11 cells that had either been incubated for 16 h with EGF (+) or left untreated (-) were treated 15 min with lactogenic hormones (DIP; +) or left untreated (-) before lysis. PTP-PEST was immunoprecipitated and analyzed either by in in-gel PTP assay (upper panel) as described in Fig. 3Go or by Western analysis for PTP-PEST (lower panel). B, Phosphatase activity ({square}) and protein level ({blacksquare}) of PTP-PEST shown in A were quantified using the ImageQuant Software (Molecular Dynamics, Inc.). The graph shows the fold induction relative to untreated control. The values represent the average ± SD of three experiments. C, PTP-PEST was immunodepleted from whole cell lysates by three rounds of immunoprecipitation (lanes 1–6); as a control, nonimmune serum was used for immunoprecipitation (lanes 7 and 8). Pellets (P) and supernatants (SN) were analyzed by an in-gel phosphatase assay.

 
To examine whether PTP-PEST was the only active PTP migrating at 125 kDa, PTP-PEST was immunodepleted from lysates of EGF-treated HC11 cells. The supernatant from the first PTP-PEST immunoprecipitation (Fig. 4CGo, lane 2) had low levels of activity (not visible in this exposure). After two rounds of immunoprecipitation, PTP-PEST was depleted from the lysates (Fig. 4CGo, lane 5), and there was no detectable PTP activity in the supernatants (Fig. 4CGo, lanes 4 and 6). The supernatants of extracts precleared with nonimmune serum showed high levels of activity at 125 kDa (Fig. 4CGo, lane 8). Thus, PTP-PEST is the only active PTP migrating at 125 kDa.

PTP-PEST Is Complexed with JAK2
Since PRL-stimulated JAK2 is negatively regulated by EGF treatment of the HC11 cells, we next examined complexes of JAK2 for associated PTP-PEST. JAK2 was immunoprecipitated from lysates of HC11 cells, which had been treated with EGF and/or the lactogenic hormones, and the immunoprecipitates were analyzed for complexed PTP-PEST by a Western blotting analysis (Fig. 5AGo, upper panel) and for PTP-PEST activity in the in-gel PTP assay (Fig. 5BGo, lower panel). We detected PTP-PEST in the JAK2 immunoprecipitates from each of the cellular lysates regardless of culturing conditions. Furthermore, there was 2-fold more PTP-PEST protein, and activity in the JAK2 immunoprecipitates from lysates of EGF-treated cultures compared with control or lactogenic hormone-treated cultures (Fig. 5Go, A and B, lanes 2 and 4; quantified in Fig. 5DGo). An in-gel assay using total cellular lysates from the same experiment is shown in the top panel of Fig. 5BGo. A constitutive complex of JAK2 and PTP-PEST was also found by performing the reverse experiment, where JAK2 was detected in immunoprecipitates of PTP-PEST (Fig. 5CGo, upper panel). In the immunoprecipitates from EGF-treated cells, which contained more PTP-PEST, there was more associated JAK2.



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Figure 5. JAK2 and PTP-PEST Are Constitutively Associated in HC11 Cells

Competent cultures of HC11 cells that had either been incubated for 16 h with EGF (+) or left untreated (-) were treated for 15 min with lactogenic hormones (DIP; +) or left untreated (-) before lysis. A, Immunoprecipitates of JAK2 were probed by Western analysis for associated PTP-PEST (upper panel). The filter was stripped and reprobed for JAK2 (lower panel). B, JAK2 was immunoprecipitated and associated PTP-PEST activity was analyzed by an in-gel PTP assay (lower panel) as described in Fig. 3Go. The upper panel shows the activity of the whole cell extracts. C, Immunoprecipitates of PTP-PEST were probed by Western analysis for associated JAK2 (upper panel). The filter was stripped and reprobed for PTP-PEST (lower panel). D, ImageQuant software (Molecular Dynamics, Inc.) was used to quantify the total activity of PTP-PEST (), JAK2-associated PTP-PEST activity ({square}) from B, and JAK2-associated PTP-PEST protein ({blacksquare}) from A. The values represent the average fold induction of two independent experiments. E, PTP-PEST or JAK2 were immunodepleted by three rounds of immunoprecipitation with specific antibodies, and control (pre-IP) and depleted lysates were compared in Western blots for PTP-PEST (lower panel) or JAK2 (upper panel). Nonimmune (ni) serum was used as a control (two left panels). To determine the quantitative amounts of PTP-PEST and JAK2 associated with each other, Western blots of three depletion experiments were analyzed using ImageQuant software (Molecular Dynamics, Inc.). The numbers below the blots show the averages as the percentage of protein relative to the nondepleted lysates.

 
To determine the fraction of PTP-PEST, which was associated with JAK2 and vice versa, we performed immunodepletion experiments on HC11 whole cell lysates using specific antisera for both proteins, then probed the supernatants for each protein. The results showed that 64% of JAK2 was complexed and coimmunoprecipitated with PTP-PEST; on the other hand, 54% of the cellular PTP-PEST could be coimmunoprecipitated with JAK2 (Fig. 5EGo).

EGF Slowly Inhibits Lactogenic Hormone-Induced ß-Casein Promoter Activity and JAK2/STAT5 Phosphorylation
In the preceding experiments HC11 cells were treated for 16 h with EGF. We next examined the kinetics of EGF’s effect on the JAK2/STAT5 pathway using two approaches. First, we examined ß-casein promoter activity using HC11 cells stably transfected with a rat ß-casein promoter-luciferase construct, HC11-Lux cells (26). Multiple transcription factors, including STAT5 and the GR, cooperate to induce ß-casein transcription (27). For HC11 cells to become transcriptionally responsive to lactogenic hormones they must be grown to and maintained at confluence for a few days, when they are referred to as competent cultures (2, 28). Treatment of control cultures of competent HC11-Lux cells for 4 h with lactogenic hormones results in an approximately 4-fold induction of luciferase activity (29). In the following experiment competent HC11-Lux cells were treated for various times with EGF, and 4 h before harvesting the lactogenic hormone mix was added. Control cultures and cultures treated 1 h with EGF displayed essentially the same luciferase activity in response to lactogenic hormones (Fig. 6Go, last two pairs of bars). The negative effect of EGF on transcriptional activation is relatively slow. Starting from 2 h of EGF treatment, there was a gradual decrease in hormonal induction of luciferase activity, which was maximal from 8 h onward (Fig. 6Go).



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Figure 6. Kinetics of EGF Inhibition of Lactogenic Hormone-Induced ß-Casein Promoter Activity

Competent cultures of HC11 cells stably transfected with a ß-casein promoter luciferase expression plasmid (HC11-Lux cells) were induced for 4 h with lactogenic hormones (DIP) before lysis. To examine the kinetics of EGF’s effect on ß-casein promoter activity, 10 ng/ml EGF were added to the cells for the indicated times before lysis. Promoter activity was measured in triplicate using total cell lysates and the luciferase assay system (Promega Corp.). In the last lane the activity of control cultures that were not treated with EGF is shown.

 
To directly examine the kinetics of EGF inhibition on JAK2/STAT5 activation, we next determined the phosphotyrosine content of JAK2 and STAT5 activity, using phospho-STAT5 antiserum, in HC11 cells pretreated for various times with EGF, then stimulated for 15 min with lactogenic hormones. There was a gradual decrease in the phosphotyrosine content of both JAK2 and STAT5, which was not evident at 4 h, but was decreased by approximately 40% and 75% after, respectively, 8 and 16 h of EGF treatment (Fig. 7AGo; quantified in Fig. 7BGo). These results agree fairly well with the kinetics of the decrease in ß-casein promoter activity observed in the previous experiment, although it should be noted that the luciferase activity was clearly decreased by 2 h whereas we generally begin to see decreased levels of phosphorylation on JAK2 and STAT5 after more than 4 h of EGF treatment. Considering the multiple transcription factors that cooperate to induce ß-casein transcription (27), it is possible that EGF has additional targets on this promoter.



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Figure 7. Kinetics of the EGF Effect on JAK/STAT Activity and PTP-PEST Induction

A, Competent cultures of HC11 cells were treated for the indicated times with EGF before treatment for 15 min with lactogenic hormones (DIP). In the upper panels JAK2 immunoprecipitates were probed in a Western analysis with a phosphotyrosine-specific monoclonal antibody, then the filter was stripped and reprobed for JAK2. In the lower panels a Western analysis for STAT5a and phospho-STAT5 was performed. B, Phosphorylation of JAK2 and STAT5 from two independent experiments, as described in A, were quantified using ImageQuant software and normalized to the protein levels. The graph represents the average phosphorylation of JAK2 and STAT5 relative to the DIP control. C, Competent HC11 were treated with 10 ng/ml EGF for the indicated times. Whole cell lysates were prepared and analyzed by Western blotting with PTP-PEST-specific antiserum (upper panel) or by an in-gel PTP assay (lower panel) as described in Fig. 3Go. D, The activity and protein level of PTP-PEST from two separate experiments were quantified using ImageQuant software. The graph shows the averages for PTP-PEST protein and activity as a percentage relative to the control value.

 
EGF Inhibition of JAK2/STAT5 Activation Requires de Novo RNA Synthesis
The results in Figs. 6Go and 7Go show that the effect of EGF on JAK2/STAT5 phosphorylation and transcriptional induction is slow. Furthermore, it has previously been demonstrated that EGF blocks lactogenic hormone-induced STAT5 DNA binding with similar, slow kinetics (30). Taken together the results suggest that the negative effect of EGF is indirect, requiring de novo RNA and protein synthesis. To test this, cells were pretreated with the RNA synthesis inhibitor actinomycin D (ActD), EGF was added to the HC11 cultures, and the lactogenic hormones were added to the medium for the final 15 min. In these experiments EGF was added for 5 h because, as shown in Fig. 8AGo, there was only a slight loss of STAT5 compared with a strong decrease in 16-h ActD-treated cultures (not shown). Quantification of the Western blots in Fig. 8AGo revealed that in EGF-treated cultures phospho-STAT5 was 36% of the control level, whereas in ActD-pretreated cells phospho-STAT5 remained at the control level (114% vs. 100%; Fig. 8AGo).



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Figure 8. EGF Inhibition of Lactogenic Hormone-Induced JAK2/STAT5 Activation Requires de Novo RNA Synthesis

Competent cultures of HC11 cells were treated for 5 h with EGF (+), lactogenic hormones (DIP) were added for 15 min, and lysates were prepared. Where indicated ActD was added 90 min before EGF addition to inhibit de novo RNA synthesis. A, Western analysis for STAT5 activation was performed using phospho-STAT5-specific antibodies (upper panel). The lower panel shows a Western blot for total STAT5a. Note that after 6.5 h of ActD treatment there was a slight decrease in the level of STAT5 in the cells. STAT5 phosphorylation and protein level were quantified using ImageQuant software. STAT5 phosphorylation levels after normalization to protein are shown below the blots as the percentage of tyrosine phosphorylation relative to the DIP control and represent the average of two independent experiments. B, Western analysis for PTP-PEST was performed using specific antibodies (upper panel). The lower panel shows the in-gel activity of PTP-PEST. C, The activity and protein level of PTP-PEST from three independent experiments, as shown in B, were quantified using ImageQuant software. The graph shows the average ± SD of the activity ({square}) and protein level ({blacksquare}) relative to the nontreated control.

 
The results presented above show that EGF stimulates the transcription of a gene(s) in HC11 cells that is necessary to mediate negative regulation of the JAK/STAT pathway. One possibility is PTP-PEST itself, as increased amounts of this protein were observed in EGF-treated HC11 cells (Figs. 4Go and 5Go). A Western blotting analysis revealed that the level of PTP-PEST gradually increased after EGF addition (Fig. 7CGo, upper panel). Quantification of the results revealed that after 4 h of treatment there was a minimal increase in PTP-PEST protein and activity (Fig. 7DGo), both of which were further increased by 1.7- and 2.2-fold in cultures treated for, respectively, 8 and 16 h with EGF. Furthermore, in cells pretreated with ActD, the EGF-induced increase in PTP-PEST protein and activity was blocked (Fig. 8BGo; quantified in Fig. 8CGo), suggesting that PTP-PEST could be an ActD-sensitive EGF target responsible for negative regulation of JAK2.

Overexpression of PTP-PEST in HC11 Cells Is Not Sufficient to Inhibit JAK2 Activity
The slow effects of EGF on both JAK2 inhibition and on PTP-PEST induction prompted us to directly ask whether overexpression of PTP-PEST inhibits JAK2. To this end PTP-PEST was transiently overexpressed in competent HC11 cultures. The adaptor protein p130cas, which has previously been identified as a PTP-PEST substrate (31, 32), was also examined in these experiments. In HC11 cultures with elevated PTP-PEST levels resulting either from long-term EGF treatment (Fig. 9AGo) or from transient overexpression (Fig. 9BGo), the phosphotyrosine content of p130cas was dramatically reduced compared with the level in control cultures (Fig. 9Go, A and B, lanes 3 vs. lanes 1 and 2). In contrast, transient overexpression of PTP-PEST did not have a significant effect on lactogenic hormone-induced STAT5 phosphorylation [Fig. 9CGo (a), lane 4] or JAK2 phosphorylation [Fig. 9CGo (b), lane 4]. In the same experiment, HC11 cells transfected with the control vector and treated for 16 h with EGF failed to respond to lactogenic hormones, displaying essentially no phosphorylation of STAT5 [Fig. 9CGo (a), lane 3] and JAK2 [Fig. 9CGo (b), lane 3]. The level of PTP-PEST expression and activity was similar in both cultures [Fig. 9CGo (c and d), lanes 3 and 4]. Importantly, there was an increase in the level of PTP-PEST complexed with JAK2 in both cultures [Fig. 9CGo (e), lanes 3 and 4], ruling out the possibility that ectopically overexpressed PTP-PEST was not associated with JAK2. Thus, in HC11 cells, p130cas was sensitive to high levels of PTP-PEST expression either induced by long-term EGF treatment or resulting from transient overexpression. In striking contrast, activity of the JAK/STAT pathway was only blocked in the EGF-treated cultures.



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Figure 9. Transient Overexpression of PTP-PEST in HC11 Cells

A, p130Cas was immunoprecipitated from lysates of HC11 cells that had either been treated 16 h with EGF (+) or left untreated (-). The immunoprecipitates were probed for phosphotyrosine content (upper panel) and reprobed for p130Cas (lower panel). B and C, HC11 were transiently transfected with PTP-PEST expression vector or empty control vector, pcDNA3.1, and incubated for an additional 2 d to allow for competency. B, p130Cas was immunoprecipitated, probed for phosphotyrosine, and reprobed for p130Cas. Expression of PTP-PEST was examined by Western blot analysis with anti-PTP-PEST antibodies. C, Lower panel, Where indicated, EGF was included for 16 h (+) in the medium of the transiently transfected HC11 cultures, and the cells were treated for 15 min with lactogenic hormones (+DIP) before lysis. a and b, Western analysis was performed using phospho-specific antibodies for STAT5 (a) and JAK2 (b). c, Expression of PTP-PEST was examined by Western blot analysis with anti-PTP-PEST antibodies. e, JAK2 was immunoprecipitated, and associated PTP-PEST activity was analyzed by in in-gel phosphatase assay with [32P]poly-(Glu:Tyr) as the gel-embedded substrate. d, The assay of total lysates.

 
PTP-PEST-Containing Lysates from EGF-Treated HC11 Cells Dephosphorylate JAK2 More Efficiently than Lysates from Control Cells
The previous experiment shows that ectopically overexpressed PTP-PEST complexed with JAK2; however, it had essentially no effect on the in vivo activity of the kinase. These results suggest that EGF induces another protein that mediates the negative regulation of JAK2. To experimentally address this, we developed an in vitro JAK2 phosphatase assay, as described in the following experiment. Lysates of HC11 cells treated for various times with EGF and precleared of JAK2 were used as the source of PTP-PEST (Fig. 10AGo). To show specificity, PTP-PEST was immunodepleted from the indicated extracts (Fig. 10AGo, lanes 2 and 6). Immunoprecipitates of activated JAK2, prepared from lactogenic hormone-treated HC11 cells, were used as the in vitro substrate. It should be noted that we employed a JAK2-specific antiserum that does not coimmunoprecipitate PTP-PEST for preparation of the substrate. JAK2 was incubated with the HC11 cell lysates, as described in Materials and Methods, and its phosphorylation was determined by a Western analysis (Fig. 10BGo). The level of phosphotyrosine in JAK2 immunoprecipitates incubated only with lysis buffer served as an input control (Fig. 10BGo, lane 1). JAK2 incubated with lysates from non-EGF-treated HC11 cells had 52% less phosphotyrosine compared with the control (Fig. 10Go, B and C, lane 3 vs. lane 1). PTP-PEST was responsible for this, since immunodepleted lysates did not dephosphorylate JAK2 (Fig. 10Go, B and C, lane 2). Importantly, the lysates prepared from EGF-treated cultures were more active toward JAK2 then the control lysate (Fig. 10Go, B and C, lanes 4 and 5 vs. lane 3). In comparison to phosphorylation after incubation with the control lysate (48% taken as 100%), JAK2 showed 52% and 87% decreases in phosphotyrosine after treatment with lysates from, respectively, 8- and 16-h EGF-treated cultures. Immunodepletion of PTP-PEST from the lysates prepared from EGF-treated cultures also restored JAK2 phosphorylation (Fig. 10Go, B and C, lane 6), although the level reached only 80% of the control value. Finally, the addition of orthovanadate to the in vitro assay reversed the effects of PTP-PEST (Fig. 10Go, B and C, lane 7). Taken together the results show that JAK2 is a PTP-PEST substrate. Moreover, they support our working model that in HC11 cells EGF slowly induces the expression of a protein that mediates the dephosphorylation of PRL-activated JAK2 by PTP-PEST (Fig. 11Go). The formal proof for this model will depend upon our identification of this protein. Furthermore, depleting HC11 cells of PTP-PEST, for example via RNA-mediated reduction (33) of PTP-PEST mRNA, will also be required to prove that PTP-PEST is the responsible PTP.



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Figure 10. PTP-PEST Dephosphorylates JAK2

The source of PTP-PEST for the in vitro assay was HC11 whole cell lysates made from cultures treated for the indicated times with EGF. These were immunodepleted of JAK2, and where indicated PTP-PEST was also immunodepleted (lanes 2 and 6) or 2 mM orthovanadate was added (lane 7). PTP-PEST protein level and activity in the lysates are shown in A. B, Active JAK2, the substrate in the in vitro phosphatase assay, was immunoprecipitated from HC11 whole cell lysates prepared from competent cultures treated for 15 min with lactogenic hormones. [PTP-PEST is not coimmunoprecipitated with the JAK2 antiserum (SC-278) used in this experiment.] Immune complexes were collected with protein A-Sepharose, washed, and used as a substrate in the vitro dephosphorylation assay, where they were treated with the lysates shown in A. In lane 1, JAK2 complexes were incubated with phosphatase buffer only. Phosphatase reactions were stopped by washing the immune complexes and boiling in sample buffer. JAK2 immunoprecipitates were Western blotted and probed for phosphotyrosine with a specific monoclonal antibody (upper panel). After stripping, the membrane was reprobed with JAK2 antiserum (lower panel). C, The relative phosphorylation level of JAK2 from two independent experiments was determined using ImageQuant software, and the averages are shown as a percentage relative to the buffer-treated control. {square}, JAK2 dephosphorylation using PEST-depleted lysates.

 


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Figure 11. Working Model for EGF-Induced Inhibition of PRL Signaling in HC11 Mammary Epithelial Cells

A, PRL-induced activation of the JAK-STAT pathway promotes transcription of milk protein genes and mammary differentiation. PTP-PEST, which is constitutively associated with JAK2, does not block JAK2/STAT5 activation. B, Transient overexpression of PTP-PEST in HC11 leads to an increase in the PTP-PEST/JAK2 association; however, PRL induction of JAK2 activity is essentially normal. C, EGF treatment of HC11 cells leads to an increase in PTP-PEST expression and PTP-PEST JAK2 association. EGF also induces the expression of a putative mediator protein X, which we propose is needed to modify the complex, allowing PTP-PEST to efficiently target JAK2 and down-regulate the response of the cells to PRL-induced differentiation.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Upon ligand binding and cytokine receptor dimerization, the associated JAK kinases are activated and phosphorylate-specific tyrosine residues in the STATs leading to their dimerization, nuclear translocation, and DNA binding (34) (35). In comparison to stimulation of the JAK/STAT pathway, processes leading to its down-regulation are less well understood. The fact that continuous JAK activity is essential for sustained STAT phosphorylation (36) and the transient nature of this phosphorylation suggest that tyrosine dephosphorylation plays an important role in negative regulation of this pathway. However, the PTPs and their specific targets are not well described (37). The results presented here suggest that PTP-PEST plays a novel role in down-regulation of PRL signaling in mammary epithelial cells. Our results imply that EGF, which we have previously shown to inhibit PRL-induced STAT5 activation (7, 8), exerts its effects via PTP-PEST down-regulation of JAK2 kinase activity. Furthermore, we demonstrate for the first time that there is constitutive PTP-PEST/JAK2 association in intact cells and that in vitro PTP-PEST dephosphorylates JAK2. The effects of EGF on HC11 cells are 2-fold. EGF stimulates PTP-PEST expression, leading to an increase in the PTP-PEST/JAK2 complex. More importantly, EGF appears to induce an as yet unknown protein, which has a permissive effect on PTP-PEST, allowing it to target and inhibit JAK2 in the PRLR complex. Our current working model is shown in Fig. 11Go.

Results from the in vitro phosphatase assay revealed that JAK2 is a PTP-PEST substrate. Importantly, the extent of JAK2 dephosphorylation increased when lysates were prepared from EGF-treated cultures, supporting our model of an EGF-induced mediator protein. We have, of course, considered alternative explanations for the results, the simplest one being that EGF induces the expression of another PTP that down-regulates JAK2 activity. Although impossible to completely rule out, there is little evidence to support this possibility. Despite the fact there are multiple PTPs in the HC11 lysates, the in vitro assay shows that JAK2 is particularly sensitive to PTP-PEST. However, we have noted that PTP-PEST immunodepletion from lysates of EGF-treated HC11 cells restored the phosphotyrosine content of JAK2 to only 80% of the control level, compared with 100% restoration using PTP-PEST immunodepleted control lysates (Fig. 10Go). Thus, it is possible that additional PTPs are induced by EGF treatment of HC11 cells; however, they were not detected in the in gel PTP assay (Fig. 3Go and data not shown). The facts that there was a specific, constitutive association of JAK2 and PTP-PEST in HC11 cells and that additional PTP-PEST, either induced endogenously by EGF or after transient transfections, became complexed with JAK2 suggest that the association of the two proteins is biologically relevant. Thus, we favor the hypothesis that an EGF- induced protein is required to mediate the effect of PTP-PEST on JAK2. Future experiments will be aimed at identifying novel JAK2-complexed proteins that augment the ability of PTP-PEST to dephosphorylate the kinase.

The results of the experiments using orthovanadate allow us to draw some general conclusions about control of JAK2 in mammary cells. First, treatment of HC11 cells with the inhibitor had no effect on JAK2 activity (Fig. 2CGo) or STAT5 DNA binding (Fig. 1Go), in control or lactogenic hormone-treated cultures. This suggests that the kinase is not held in check by the complexed PTP-PEST or any other PTP and shows that JAK2 activity is strictly regulated by PRL binding to its receptor. Even after JAK2 activation PTP-PEST did not negatively regulate the kinase, as orthovanadate treatment of HC11 cultures had no effect on lactogenic hormone-mediated JAK2 stimulation. Only in EGF-treated cultures could orthovanadate treatment restore JAK2 activity and STAT5 DNA binding to their normal levels. This contrasts with the in vitro assay where we observed PTP-PEST-mediated JAK2 dephosphorylation to 48% of the control level when lysates were prepared from non-EGF-treated HC11 cells, perhaps because some restraints were lost during preparation of the lysates. Taken together, the results suggest that in intact cells the activity of PTP-PEST toward JAK2 is kept tightly controlled. Only after EGF treatment and presumably association of a mediating protein with the complex is PTP-PEST able to dephosphorylate JAK2.

Negative regulators of the JAK/STAT pathway have previously been described. These include PTPs as well as non-PTPs (37); of the latter the most well known is a family of proteins known as suppressors of cytokine signaling (SOCS) or cytokine-inducible SH2-containing protein (CIS). These proteins are transcriptionally induced by STATs in response to a variety of cytokines. The SOCS/CIS proteins bind either directly to JAK kinases, inhibiting their activity, or to activated receptors, thereby preventing the recruitment and phosphorylation of STATs (37). It has been shown that PRL-induced STAT activity can be down-regulated after transfection of various SOCS/CIS family members (38). Furthermore, PRL up-regulates the transcription of diverse SOCS/CIS mRNAs in the T47D breast tumor cell line (38) and in mammary glands of lactating mice (39). Thus, it is very likely that SOCS/CIS proteins have general roles in controlling the STAT5 response to lactogenic signaling in mammary cells. However, for various reasons they do not appear to be involved in the process that we describe here. SOCS/CIS have not yet been described to be up-regulated in response to EGF. More importantly, SOCS/CIS-mediated effects on cytokine signaling do not require PTP activity, nor are they sensitive to treatment with orthovanadate, which contrasts with what we have observed in our experiments.

Turning to phosphatases, specific PTPs have been implicated in the negative regulation of JAK2 and/or STAT5. CD45, a transmembrane PTP highly expressed in cells of the hemopoietic lineage, has recently been shown to dephosphorylate JAK2, leading to negative regulation of multiple cytokines (40). Overexpression of the cytosolic PTP1B in COMMA-1D cells, from which HC11 cells were originally isolated (1), led to a decrease in PRL-induced STAT5 phosphorylation (41). In these experiments JAK2 activity was not affected by PTP1B expression, suggesting that STAT5 was the direct target. The SH2 domain-containing protein tyrosine phosphatases (SHPs), SHP1 and SHP2, have also been implicated in down-regulation of cytokine receptor signaling (34). SHP1 has been reported to associate with and dephosphorylate JAK2 (42). Furthermore, in response to GH, SHP1 associates with STAT5b in livers of male rats, where it may contribute to termination of GH signaling (43). STAT5 has also been reported to serve as a SHP2 substrate in an in vitro assay and trapping substrate mutants of SHP2 complex with STAT5 (44, 45). We detected SHP1 and SHP2 activity using the in-gel PTP assay on HC11 whole cell extracts (Fig. 3AGo, asterisk). However, neither SHP1 nor SHP2 was complexed with JAK2, nor was their activity increased in EGF-treated HC11 cells. In contrast, the activity of SHP2 was increased after treatment with the cytokine IL-6 (Horsch, K., unpublished data), showing that the in-gel PTP assay is sensitive enough to see changes in activity of these PTPs. Taken together, these results suggest that neither of the SH2-containing PTPs plays a major role in deactivation of JAK2 signaling in the HC11 cells in response to lactogenic hormones or EGF.

An emerging theme in signal transduction is the cross-regulation of different classes of membrane receptors. Receptor tyrosine kinases and cytokine receptors in some instances cooperate to induce signaling pathways (44, 46). Negative regulation has also been observed, and considering the mammary gland specifically, there is a complex interaction between EGFR and PRLR. On the one hand, PRLR activation has a negative influence on EGF-induced signaling. In mammary cells continuously exposed to PRL, EGFR has been shown to be phosphorylated on specific threonine residues, a modification that had a negative effect on its kinase activity, leading to impaired EGF-induced biological responses (47). On the other hand, as shown here, continuous exposure of HC11 mammary cells to EGF dampens their response to PRL. It is interesting to consider the negative regulation mediated by PRLR on EGFR and vice versa in light of their roles in the development of the mammary gland. EGFR is expressed at all stages (48, 49) and appears to have a particularly important role in proliferation of ductal epithelial cells, as wa-2 mice, which harbor a mutation in the EGFR kinase domain, exhibit sparse ductal growth (10). PRLR does not have a major role in the proliferative phase; however, it plays an essential role in differentiation of the gland into a milk-producing organ (50). The negative effects of EGF on PRL signaling, which we have described, here might be a part of a more general control of differentiation-inducing stimuli during the normal proliferative stage of development. During the rapid growth of the gland that occurs at sexual maturity and continues during pregnancy, there appear to be numerous mechanisms inhibiting differentiation and milk protein gene expression. These range from negative regulatory transcription factors (4, 27, 51) that block expression of differentiation specific genes to the mechanism we describe here whereby EGF directly impinges upon one of the major differentiation-inducing signaling molecules, JAK2.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
The antibodies used were EGFR-specific rabbit polyclonal antibody SC-03 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); STAT5a and STAT5b polyclonal antisera (29); phospho-STAT5-specific monoclonal antibody, which recognizes both isoforms (Upstate Biotechnology, Inc., Lake Placid, NY) (52); JAK2-specific polyclonal antibodies SC-278 (Santa Cruz Biotechnology, Inc.) and 06-255 (Upstate Biotechnology, Inc.), phospho-JAK2-specific rabbit polyclonal antiserum (44-426, BioSource Technologies, Inc., Camarillo, CA) which recognizes Tyr1007/1008 in the activation site; PTP-PEST-specific polyclonal antiserum (53); phosphotyrosine-specific monoclonal antibody (Upstate Biotechnology, Inc.); and p130cas polyclonal antiserum SC-860 (Santa Cruz Biotechnology, Inc.). PRLR-specific polyclonal antiserum, generated by immunizing rabbits with a peptide encompassing amino acids 466–478 of mouse PRLR, was provided by Dr. Charles Streuli (Manchester, UK). The growth factors and hormones used were recombinant human EGF, dexamethasone, insulin, and ovine PRL (all from Sigma, St. Louis, MO). The inhibitors used were ActD (Sigma) and sodium orthovanadate (Sigma).

Cell Culture and Transfections
HC11 mammary epithelial cells were grown to confluence and maintained for 2 d in growth medium (RPMI 1640 supplemented with 10% FCS, 10 ng/ml EGF, and 5 µg/ml insulin). These are referred to as competent cultures (28). To induce differentiation, EGF was removed from the medium a minimum of 24 h before addition of the lactogenic hormone mix (DIP). Transient transfections were performed as follows. HC11 cells were grown to 70% confluence, and control empty vector or a plasmid encoding the PTP-PEST cDNA (53) was introduced into cells using the Fugene transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s protocol. Two days after transfection the cells were confluent, EGF was removed from the medium for 24 h, then the cultures were treated for 15 min with lactogenic hormones, and lysates were prepared in Nonidet P-40 buffer [1% Nonidet P-40, 50 mM Tris (pH 7.5), 120 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.5 mM phenylmethylsulfonylfluoride, 5 mM sodium fluoride, 20 µg M ß-glycerophosphate, and 2 mM sodium orthovanadate].

Luciferase Assays
HC11 cells stably transfected with a ß-casein promoter luciferase construct (p-334/-1ßc-Lux) (29) were grown to and maintained for 2 d at confluence, then treated 4 h with lactogenic hormone-containing medium. To examine the kinetics of EGF’s effect on induction of luciferase activity, the cultures were incubated with 10 ng/ml EGF for various times before or after the addition of lactogenic hormones, which in all cases were present for 4 h. Luciferase activity was determined on triplicate samples using the luciferase assay system (Promega Corp., Madison, WI) as described by the manufacturer. Total light emission was measured during the first 3 sec of the reaction using a luminometer (Berthold Microlumat LB96P).

EMSA
Nuclear extracts were prepared by scraping cells into cytoplasmic extraction buffer [10 mM KCl, 20 mM HEPES (pH 7.0), 1 mM MgCl2, 0.1% Triton X-100, 20% glycerol, 0.1 M EGTA, 0.5 mM DTT, 2 mM sodium orthovanadate, 50 µM ß-glycerophosphate, 50 mM sodium fluoride, 2 mM phenylmethylsulfonylfluoride, 5 µg/ml leupeptin, and 5 µg/ml aprotinin] and shearing with 20 strokes using a Dounce homogenizer (Wheaton, pestle B, Kontes Co., Vineland, NJ). Nuclei were pelleted by centrifugation at 800 x g for 5 min and then treated with nuclear extraction buffer (cytoplasmic extraction buffer and 300 mM NaCl) for 30 min on ice. Extracts were clarified by centrifugation for 15 min at 16,000 x g. EMSAs were performed by incubating 10 µg nuclear protein extract with the STAT5 DNA-binding site from the rat ß-casein promoter (5'-AGATTTCTAGGAATTCA ATCC-3') (5) for 30 min on ice in 20 µl EMSA buffer [10 mM HEPES (pH 7.6), 2 mM NaH2PO4, 0.25 mM EDTA, 1 mM DTT, 5 mM MgCl2, 80 mM KCl, 2% glycerol, and 100 µg/ml poly(dI-dC)]. Specific binding was analyzed on a 4% native polyacrylamide gel, prerun for 2 h at 200 V in 0.25x TBE [22.5 mM Tris borate (pH 8.0) and 0.5 mM EDTA]. The samples were loaded and electrophoresed for 1 h at 200 V, and the gels were dried and autoradiographed.

Immunoprecipitation and Western Blot Analysis
Whole cell lysates were obtained by solubilizing cells in Nonidet P-40 buffer [1% Nonidet P-40, 50 mM Tris (pH 7.5), 120 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM DTT, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.5 mM phenylmethylsulfonylfluoride, 5 mM sodium fluoride, 20 µM ß-glycerophosphate, and 2 mM sodium orthovanadate] for 5 min on ice. The lysates were clarified by centrifugation at 16,000 x g for 15 min. For immunoprecipitations, equal amounts of protein were incubated with specific antibodies for 1–2 h on ice. Immune complexes were collected with protein A-Sepharose (Sigma) by rotating the complexes for 30 min at 4 C and washing three times with lysis buffer and once with TNE buffer (50 mM Tris, 140 mM NaCl, and 5 mM EDTA). Immunoprecipitated proteins were boiled in sample buffer and analyzed by SDS-PAGE. Immunodepletion was performed by two or three consecutive incubations of lysates with the specific antiserum. Proteins were electroblotted onto polyvinylidene difluoride membranes (Roche Molecular Biochemicals) and detected with specific antiserum after blocking the filters with 10% horse serum (Life Technologies, Inc., Grand Island, NY) in TTBS [50 mM Tris (pH 7.5), 150 mM NaCl, and 0.05% Tween 20]. Proteins were visualized with peroxidase-coupled secondary antibody using the ECL detection system (Amersham Pharmacia Biotech). For reprobing, membranes were stripped in 62.5 mM Tris (pH 6.8), 2% SDS, and 100 mM ß-mercaptoethanol for 30 min at 60 C.

In Vitro JAK2 Kinase Assay
The cells were lysed in Nonidet P-40 extraction buffer, and 1.5-mg aliquots were precleared with protein A-Sepharose for 30 min on ice, followed by incubation with a JAK2-specific polyclonal antibody for 2 h on ice. Immune complexes were collected with protein A-Sepharose by rotation for 30 min at 4 C and washed four times with cold lysis buffer and twice with kinase buffer [50 mM Tris (pH 7.5), 0.1% Nonidet P-40, 100 mM NaCl, 10 mM MgCl2, 5 mM MnCl, 100 µM sodium orthovanadate, and 1 mM DTT]. Kinase buffer (100 µl) with or without 15 µM ATP was added to the immunoprecipitates, which were incubated for 30 min at 37 C on a Thermo-Shake (Eppendorf, Hamburg, Germany) (22, 23). The kinase reaction was stopped by washing with cold lysis buffer and boiling the complexes in sample buffer. Immunoprecipitated proteins were subjected to SDS-PAGE, blotted onto polyvinylidene difluoride membranes, and probed with a phosphotyrosine-specific monoclonal antibody. After stripping, membranes were reprobed with JAK2-specific antiserum.

In-Gel Phosphatase Assay
We employed an in-gel PTP assay essentially as described previously (22). For detection of PTP activity, SDS-polyacrylamide gels containing 2 x 105 cpm/ml 32P-labeled substrate were prepared. The substrate (poly-[glutamic-acid:tyrosine]; 20,000–50,000 kDa; Sigma) was dissolved in kinase buffer [30 mM MgCl2, 1 mM MnCl2, 1 mM sodium vanadate, 10 mM DTT, 0.05% Triton X-100, 50 mM imidazole (pH 7.2), and 0.2 mM ATP] and incubated with [32P]ATP (Amersham Pharmacia Biotech, Piscataway, NJ) and recombinant glutathione-S-transferase-c-Src (provided by Dr. F. Boehmer, University of Jena, Jena, Germany) for 18 h by rotation at room temperature. The phosphatase substrate was purified via a Sepharose G-50 column (Amersham Pharmacia Biotech). Nonidet P-40 cell lysates (50 µg) were electrophoresed, and in-gel PTP activity was visualized as follows. The gels were sequentially shaken for the indicated times at room temperature in 250 ml of the following buffers: 90 min in PB1 [50 mM Tris (pH 8.0) and 20% isopropanol); twice for 30 min each time in PB2 [50 mM Tris (pH 8.0) and 0.3% ß-mercaptoethanol]; 90 min in PB3 [50 mM Tris (pH 8.0), 0.3% ß-mercaptoethanol, 6 M guanidine hydrochloride, and 1 mM EDTA], three times for 30 min each time in PB4 [50 mM Tris (pH 8.0), 0.3% ß-mercaptoethanol, 1 mM EDTA, and 0.04% Tween 40], and 15 h in PB5 [50 mM 2-morpholinoethanesulfonic acid (pH 6.0), 0.3% ß-mercaptoethanol, 1 mM EDTA, 0.04% Tween 40, and 4 mM DTT]. After these incubations, gels were stained with Coomassie brilliant blue, destained in 40% ethanol/10% acetic acid, dried, and analyzed using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

In Vitro PTP Assay
JAK2 was immunoprecipitated from 500 µg Nonidet P-40 lysates prepared from competent cultures of HC11 cells treated for 15 min with lactogenic hormones. The JAK2 antiserum used, Santa Cruz SC-278, does not coimmunoprecipitate PTP-PEST. Immune complexes were collected with protein A-Sepharose, washed with inhibitor-free Nonidet P-40 buffer (Nonidet P-40 buffer lacking phenylmethylsulfonylfluoride, NaF, sodium vanadate, and ß-glycerophosphate), and used as substrate in the in vitro dephosphorylation assay. Lysates were prepared in inhibitor-free Nonidet P-40 buffer from competent cultures of HC11 cells, either left untreated or treated for various times with 10 ng/ml EGF. Lysate (500 µg) from which JAK2 was immunodepleted was used as the source of PTP-PEST for the in vitro assays. As controls, lysates from which PTP-PEST had also been immunodepleted or lysates containing 2 mM sodium orthovanadate were employed. For in vitro dephosphorylation, 300 µl lysate plus 300 µl phosphatase buffer [50 mM 2-morpholinoethanesulfonic acid (pH 6.0), 0.3% ß-mercaptoethanol, 1 mM EDTA, 0.04% Tween 40, and 4 mM DTT] were added to the JAK2-protein A-Sepharose complexes, and these were rotated at 4 C for 30 min. To determine the initial JAK2 phosphotyrosine content, the JAK2-protein A-Sepharose complexes were rotated with 300 µl Nonidet P-40 lysis buffer plus 300 µl phosphatase buffer. The reaction was terminated by pelleting the JAK2 immunocomplexes and washing the pellets three times with Nonidet P-40 buffer containing phosphatase inhibitors and once with TNE. Immunoprecipitated JAK2 was released by boiling in sample buffer, subjected to SDS-PAGE, and blotted onto polyvinylidene difluoride membranes, which were probed with a phosphotyrosine-specific monoclonal antibody. After stripping the membrane, the filters were reprobed with JAK2-specific antiserum.


    ACKNOWLEDGMENTS
 
We thank C. Streuli for the PRLR antiserum, F. Boehmer for Src kinase, and M. Wartmann for initial results with the HC11-Lux cells. We thank K. Burridge, T. Holbro, C. Streuli, A. Badache, B. Hemmings, and the members of the Hynes laboratory for helpful suggestions and discussions.


    FOOTNOTES
 
This work was supported by the Novartis Research Foundation.

Abbreviations: ActD, Actinomycin D; CIS, cytokine-inducible SH2-containing protein; DIP, lactogenic hormone mixture (10-6 M dexamethasone, 5 µg/ml insulin, and 5 µg/ml ovine PRL); DTT, dithiothreitol; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; JAK, Janus kinase; PEST, proline glutamine serine threonine-rich sequence; PRLR, PRL receptor; SOCS, suppressors of cytokine signaling; SHP, SH2 domain-containing protein tyrosine phosphatase; STAT, signal transducer and activator of transcription.

Received for publication March 5, 2001. Accepted for publication August 27, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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