Extracellular Signal-Regulated Kinase (ERK) Interacts with Signal Transducer and Activator of Transcription (STAT) 5a

Tony J. Pircher, Hanne Petersen, Jan-ke Gustafsson and Lars-Arne Haldosén

Department of Medical Nutrition Karolinska Institute, Novum S-141 86 Huddinge, Sweden


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Serine phosphorylation of signal transducers and activators of transcription (STAT) 1 and 3 modulates their DNA-binding capacity and/or transcriptional activity. Earlier we suggested that STAT5a functional capacity could be influenced by the mitogen-activated protein kinase (MAPK) pathway. In the present study, we have analyzed the interactions between STAT5a and the MAPKs, extracellular signal-regulated kinases ERK1 and ERK2. GH treatment of Chinese hamster ovary cells stably transfected with the GH receptor (CHOA cells) led to rapid and transient activation of both STAT5a and ERK1 and ERK2. Pretreatment of cells with colchicine, which inhibits tubulin polymerization, did not inhibit STAT5a translocation to the nucleus and ERK1/2 activation. In vitro precipitation with a glutathione-S-transferase-fusion protein containing the C-terminal transactivation domain of STAT5a showed GH-regulated association of ERK1/2 with the fusion protein, while this was not seen when serine 780 in STAT5a was changed to alanine. In vitro phosphorylation of the glutathione-S-transferase-fusion proteins using active ERK only worked when the fusion protein contained wild-type STAT5a sequence. The same experiment, performed with full-length wild-type STAT5a and the corresponding S780A mutant, showed that serine 780 is the only substrate in full-length STAT5a for active ERK. In coimmunoprecipitation experiments, larger amounts of STAT5a-ERK1/2 complexes were detected in cytosol from untreated CHOA cells than in cytosol from GH-treated cells, suggesting the presence of preformed STAT5a-ERK1/2 complexes in unstimulated cells. Transfection experiments with COS cells showed that kinase-inactive ERK1 decreased GH stimulation of STAT5-regulated reporter gene expression. These observations show, for the first time, direct physical interaction between ERK and STAT5a and also clearly identify serine 780 as a target for ERK. Furthermore, it is also established that serine phosphorylation of STAT5a transactivation domain, via the MAPK pathway, is a means of modifying GH-induced transcriptional activation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Signal transducers and activators of transcription (STAT) proteins are a family of latent cytoplasmic transcriptional regulators (1, 2). Ligand binding to cytokine receptors (including receptors for GH, several interleukins, PRL, and erythropoietin) and some tyrosine kinase receptors (including epidermal growth factor receptor and platelet-derived growth factor receptor) leads to activation of STAT proteins through tyrosine phosphorylation. In the case of cytokine receptors, tyrosine phosphorylation is mediated by receptor-bound members of the Janus kinase (JAK) family (3, 4, 5). Tyrosine phosphorylation of STAT proteins is obligatory for dimerization, translocation to the nucleus, and regulation of specific genes.

The importance of serine phosphorylation of STAT proteins has recently been acknowledged. Studies have mainly focused on serine 727 in STAT1{alpha} and STAT3, which is situated in a mitogen-activated protein kinase (MAPK) consensus recognition sequence, PMSP (6, 7, 8, 9, 10). Phosphorylation of serine 727 has been shown to regulate DNA binding capacity and/or transcriptional activity of these STAT proteins. Furthermore, treatment of the human fibroblast cell line (U266) with interferon-ß (IFN-ß)-induced association of STAT1{alpha} with ERK2 (11).

We have shown earlier that GH activates JAK2/STAT5 and MAPK pathways (12, 13). We have also shown that GH activates both isoforms of STAT5, STAT5a and STAT5b (13). These isoforms differ mainly at the C-terminal end, one of the differences being the presence of a possible ERK phosphorylation site (RLSP) in STAT5a (14). Recently, we have examined the influence of MAPK signaling on the JAK2/STAT5 pathway. Pretreatment of GH-responsive cells with the MAPK kinase (MEK) inhibitor PD98059 resulted in a decreased amount of GH-induced nuclear STAT5 with DNA-binding capacity and reduced transcriptional activation of STAT5-regulated reporter gene (13), whereas GH-stimulated nuclear translocation of STAT5 was not influenced. Data indicated that STAT5a is dependent on MAPK activity for full transcriptional activity and that serine 780 of STAT5a within the putative MAPK recognition sequence is a likely target for MAPK.

In this study we have further investigated GH activation of the MAPK pathway, as well as the interaction between this pathway and the JAK2/STAT5 system. In particular, we have obtained firm evidence for protein-protein interaction between STAT5a and ERK, established that serine 780 of STAT5a is a target for ERK, and also directly correlated ERK activity with STAT5a functional capacity.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Kinetics of ERK and STAT5a Activation after GH Stimulation
Data from our previous study indicated that MAPK could influence STAT5a functional capacity (13). In our present study we have investigated whether ERK1/2 and STAT5a follow the same activation kinetics after GH stimulation and whether GH activates both ERK1 and ERK2. CHOA cells were incubated with GH for different times, and cytosol and nuclear extracts were analyzed using Western blotting with antibodies against ERK1/2, active ERK1/2, and STAT5a. The antibody against active ERK1/2 only recognizes ERK1/2 where threonine 183 and tyrosine 185 are phosphorylated. Phosphorylation of these two amino acids by MEK results in activation of ERK1/2 (15, 16, 17). In cytosolic extract, active ERK1 and 2 were detected after 4 min of GH stimulation (Fig. 1AGo, middle panel). The highest levels of active ERK1/2 were seen after 8 min of stimulation, after which the active forms slowly disappeared up until 20 min of GH stimulation. GH induced higher levels of active ERK2 than ERK1. The antiactive ERK antibody has been reported to have the same affinity for active forms of ERK1 and ERK2 (18). No significant reduction of STAT5a or ERK1/2 protein levels in cytosol (as a result of nuclear translocation) was detected upon GH stimulation (Fig. 1AGo, upper and lower panel), indicating that GH activated a minor pool of ERK1/2 and STAT5a. Analysis of nuclear extracts showed that both ERK1 and 2 were present in the nucleus in the absence of GH stimulation, while STAT5a translocated to the nucleus after GH stimulation (Fig. 1BGo, upper and lower panel). Active forms of ERK1/2 were clearly detected after 4 min of GH stimulation (Fig. 1BGo, middle panel). The highest levels of active ERK1/2 in nuclear extracts were seen after 8–12 min. The highest level of nuclear STAT5a protein was seen after 4–8 min, after which a slow decrease occurred, a pattern that resembled that of the DNA-binding capacity of nuclear STAT5, as analyzed by gel shift technique (Fig. 1CGo). From the above data it can be concluded that GH activated both ERK forms and that ERK1 and 2 and STAT5a had similar activation kinetics after GH stimulation of CHOA cells.



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Figure 1. Activation of ERK1, ERK2, and STAT5a by GH in CHOA Cells

CHOA cells, grown in serum-free medium, were treated with or without 100 nM GH for indicated times. Cytosolic extract (panel A) and nuclear extracts (B) were analyzed by Western blot technique using polyclonal antibodies against STAT5a, ERK1/2, and active MAPK. C, Nuclear extracts were analyzed with a gel shift method using a STAT5-binding oligonucleotide as a probe.

 
Activation of ERK and Nuclear Translocation of STAT5a after Depolymerization of Microtubuli
MAPKs were initially described as microtubuli-associated proteins (19, 20). Later investigations showed that MAPKs are also present in cytosolic compartments not associated with microtubuli and also in the nucleus (21, 22). We have investigated whether intact microtubuli are required for GH activation of ERK1/2 in CHOA cells (Fig. 2Go). Furthermore, we have analyzed whether translocation of STAT5a from cytosol to nucleus is dependent on intact microtubuli. CHOA cells were pretreated with or without 10 µM colchicine before treatment with GH. This concentration of colchicine has been shown to effectively depolymerize microtubuli in CHOA cells (E. L. K. Goh, Pircher, T.J. and Lobie, P.E., manuscript submitted). Cytosolic and nuclear extracts from treated cells were analyzed by Western blot using antibodies against active ERK and STAT5a. Analysis of nuclear extract indicated that depolymerization of microtubuli did not inhibit GH-induced activation of ERK1/2 and also did not inhibit nuclear translocation of STAT5a. Analysis of cytosolic extract showed no decrease in ERK1/2 activation by GH after treatment with colchicine (data not shown). Thus, intact microtubuli were not required for GH-induced activation of ERK1/2 and GH-induced nuclear translocation of STAT5a.



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Figure 2. Depolymerization of Microtubuli with Colchicine

CHOA cells, grown in serum-free medium, were pretreated for 60 min with or without 10 µM colchicine before GH treatment for different times. Nuclear extracts were analyzed with Western blot technique using polyclonal antibodies against STAT5a and active MAPK.

 
Influence of ERK on STAT5a Functional Capacity
We showed earlier that inhibition of MEK with PD98059 resulted in decreased GH-induced activation of STAT5-regulated reporter gene expression and proposed that decreased MAPK activity was responsible for this effect of PD98059 (13). In the present study we have analyzed the direct influence of ERK activity on STAT5-regulated transcription. COS cells were transfected with expression vectors for GH receptor, STAT5a, and wild-type or kinase mutant ERK1. In kinase mutant ERK1-transfected cells, GH-induced reporter gene expression was significantly reduced as compared with COS cells transfected with wild-type ERK1 (Fig. 3Go). Thus, STAT5a functional capacity was influenced by active ERK1.



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Figure 3. ERK-Dependent Expression of STAT5-Regulated Reporter Gene

COS cells were transfected with the STAT5-regulated reporter construct, SPI-GLE1-luc, and expression vectors for GHR, STAT5a, wild-type ERK1 (ERK-wt), or kinase inactive ERK1 (ERK-kin-) or control (pcDNA) and ß-galactosidase. After 12 h treatment with 100 nM hGH, cells were harvested and extracts were assayed for luciferase and ß-galactosidase activities. Values shown represent normalized luciferase activities in arbitrary units. Results represent the mean ± SD of triplicate estimations. Results presented are representative of three experiments.

 
GH-Regulated Interaction between ERK and C-Terminal Part of STAT5a
We have previously suggested that serine 780, within the C-terminal part of STAT5a, is a MAPK substrate (13). To further strengthen this notion, we produced glutathione-S-transferase (GST)-fusion proteins either containing wild-type sequence of the C-terminal 64 amino acids of STAT5a or the same 64 amino acids but with serine 780 mutated to alanine (Fig. 4AGo). Figure 4BGo shows a Coomassie-stained SDS-PAGE gel with purified GST-fusion proteins. For both wild-type and serine-mutated GST-fusion constructs, two main bands could be seen. These proteins were also detected by Western blot analysis with anti-STAT5a antibody directed against the last 18 C-terminal amino acids of STAT5a (data not shown). GST-fusion proteins were used for precipitation and in vitro phosphorylation assays. CHOA cells were treated with or without GH for 10 min. Cytosolic extracts from these cells were incubated with GST and wild-type or serine mutant C-terminal STAT5a GST fusion proteins. Precipitates were separated on SDS-PAGE and analyzed by Western blot with anti-ERK1/2 antibody (Fig. 4CGo). GST fusion proteins and, to a smaller extent, GST precipitated ERK1 in extracts from stimulated or unstimulated cells. ERK2 coprecipitated with wild-type and, to a smaller extent, with mutant GST fusion protein in extracts from stimulated or unstimulated cells. GH treatment of CHOA cells induced an increase in association of ERK1 and 2 to wild-type GST fusion protein, an effect that could not be seen with serine mutant GST fusion protein or GST. GST and GST fusion proteins were also incubated with commercially available active ERK and radiolabeled ATP. Incubations were analyzed using SDS-PAGE/autoradiography. As can be seen in Fig. 4DGo, only GST fusion protein containing wild-type C-terminal sequence of STAT5a was phosphorylated. These data suggest a GH-regulated interaction of ERK1/2 with the C-terminal part of STAT5a and that serine 780, within that part, is a target for ERK.



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Figure 4. In Vitro Interaction between GST-STAT5a and Recombinant Active MAPK

A, C-terminal STAT5a GST-fusion proteins constructed. B, Coomassie staining of SDS-PAGE gel loaded with purified GST (C) and STAT5a GST-fusion proteins (WT and SA). Two different amounts of GST-fusion proteins were loaded. C, Cytosolic extract from CHOA cells, treated with and without 100 nM GH, was precipitated with either 4 µg of GST (C), wild-type (WT), or S780A (SA) C-terminal STAT5a fusion proteins. Precipitations were analyzed by Western blot using anti-ERK1/2 antibody. D, The GST-fusion constructs were also incubated in vitro with active MAPK and ATP(32P) for 30 min at 30 C. Phosphorylated entities were detected by SDS-PAGE/autoradiography.

 
ERK Phosphorylation of Recombinant Full-Length STAT5a in Vitro
Analysis of the amino acid sequence of STAT5a showed that more possible serine/threonine phosphorylation sites exist in addition to the C-terminal MAPK phosphorylation site. To investigate whether these sites are targets for ERK and also to determine whether MAPK can phosphorylate serine 780 in the full-length protein, we studied ERK phosphorylation of full-length STAT5a in vitro. We expressed recombinant His-tagged wild-type STAT5a and serine 780 alanine mutant of STAT5a (STAT5aS780A) in insect cells using the baculovirus system (Fig. 5AGo). Western blot analysis of extracts from infected cells with anti-STAT5a antibody confirmed the expression of recombinant proteins (Fig. 5BGo). We have previously shown that recombinant MGF (STAT5) is tyrosine phosphor-ylated in insect cells by an unknown endogenous tyrosine kinase and that this MGF has DNA-binding capacity with the proper binding specificity (23). We analyzed DNA-binding capacity and specifity for recombinant wild-type and mutant STAT5a. Figure 5DGo shows the result of gel shift analysis of recombinant STAT5aS780A in which an oligonucleotide containing the STAT5 binding site from ß-casein promoter was used as probe. The recombinant mutant STAT5a had DNA-binding capacity, which was lost when insect cells were treated with the protein kinase inhibitor staurosporine (data not shown). The recombinant serine mutant STAT5a also exhibited the same DNA binding specificity as earlier determined for recombinant MGF and STAT5 from CHO cells (23, 24). Identical results were obtained when recombinant wild-type STAT5a was analyzed in the same way (data not shown). This indicates that both recombinant STAT5a forms are expressed properly folded. Full-length STAT5a and STAT5aS780A were incubated together with active ERK and radiolabeled ATP, after which the incubations were analyzed by SDS-PAGE/autoradiography. Figure 5CGo shows that only recombinant wild-type STAT5a was phosphorylated. We have also used wild-type STAT5a and serine mutant STAT5a, expressed in COS cells, in in vitro phosphorylation experiments. These experiments also showed that only wild-type STAT5a was phosphorylated (data not shown). Taken together, these experiments show that ERK phosphorylated full-length STAT5a and that serine 780 was the only MAPK phosphorylation site in STAT5a under these in vitro conditions.



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Figure 5. In Vitro Phosphorylation of Full-Length Recombinant STAT5a with Active MAPK

A, Recombinant STAT5a proteins. B, Western blot of extracts from Sf9-insect cells infected with recombinant baculovirus showing expression of wild-type STAT5a (WT) and STAT5aS780A (SA). Extract from noninfected cells was used as control (C). C, In vitro phosphorylation of recombinant baculovirus- expressed proteins with active MAPK. D, Effect of competition with GAS-like oligonucleotides on binding of recombinant STAT5aS780A to ß-casein probe. Excesses of various GAS-like oligonucleotides were used as competitors. See Materials and Methods for key.

 
Coprecipitation of STAT5a and ERK
To show interaction between ERKs and STAT5a in cells with endogenous levels of these proteins, we treated CHOA with GH for 8 min and prepared cytosolic extracts. Equal amounts of polyclonal anti-ERK1/2 or anti-ERK2 antibody were added to extracts for precipitation. Precipitates were analyzed by Western blot with antibodies against STAT5a or ERK1/2. The anti-ERK1/2 antibody precipitated ERK1 and ERK2, with ERK1 being precipitated better than ERK2 (Fig. 6AGo). It was only upon longer exposure that weak STAT5a coimmunoprecipitation could be detected in extracts from treated or untreated CHOA cells (data not shown). We have tested two different anti-ERK1/2 antibodies from different manufacturers with the same results. Both of these antibodies are directed against the C-terminal part of ERK, amino acids 339–353 and 333–367, respectively. The anti-ERK2 antibody precipitated, in our hands, repetitively both ERK1 and ERK2. This antibody is raised against full-length murine ERK2. With this antibody STAT5a coimmunoprecipitated in extracts from both treated and untreated cells. A considerably lower amount of STAT5a was coimmunoprecipitated in extract from treated cells, indicating that a complex between STAT5a and ERK1 and/or ERK2 was present in unstimulated CHOA cells that started to dissociate upon GH stimulation. This finding was further investigated by transfection of COS cells with expression vector for GH receptor and different combinations of expression vectors for wild-type or serine 780 mutant of STAT5a and hemagglutinin (HA)-tagged wild-type or kinase mutant of ERK1. COS cells were then treated with GH for 8 min. Cytosolic extract was prepared, and anti-HA antibody was added for immunoprecipitation. Precipitates were analyzed by Western blot with anti-STAT5a antibody. In Fig. 6BGo it can be seen that wild-type and serine 780 mutant of STAT5a coimmunoprecipitated with both wild-type and kinase mutant ERK1. The amount of wild-type STAT5a coimmunoprecipitated in extracts from COS cells transfected with wild-type STAT5a and wild-type ERK1 was less than in the other extracts. Thus, both STAT5a forms were complexed with both ERK1 forms, but dissociation of complex upon GH stimulation only occurred in COS cells transfected with wild-type STAT5a and wild-type ERK1.



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Figure 6. GH Modulated Co-Precipitation of STAT5a with ERK2 from CHOA Cells

A, CHOA cells, serum deprived over night, were treated with and without 100 nM GH. Cytosolic extracts were incubated with polyclonal antibody against ERK or ERK2. Antibody-bound material was precipitated with protein A-Sepharose and analyzed by Western blotting with antibodies against STAT5 and ERK1/2. B, COS cells were transfected with the expression vectors for GH receptor and STAT5a or STAT5aS780A and HA-tagged wild-type ERK1 (HA-ERK-wt) or HA-tagged kinase inactive ERK1 (HA-ERK-kin-). After 10 min of GH stimulation, cytosolic extracts were prepared. Anti-HA antibody was added to extracts, after which bound material was precipitated with protein A-Sepharose and analyzed by Western blotting using antibodies against STAT5a.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In our previous study we showed that inhibition of MEK with PD98059 before treatment of cells with GH resulted in inhibition of GH-induced MAPK activation, reduction of GH-induced STAT5 in nuclear extract with DNA binding capacity, and decreased GH-induced activation of a STAT5-regulated reporter construct (13). Experiments in that study also indicated that STAT5a, which has a MAPK recognition sequence within its C-terminal domain, could be a target for MAPK. Since this is an important issue for the understanding of GH signaling, we have specifically addressed this notion using several experimental approaches.

STAT5a and ERK1/2 coimmunoprecipitated from extracts of CHOA cells, indicating that STAT5a and ERK1/2 can complex in these cells. An unexpected finding was that a lower amount of STAT5a-ERK1/2 complex was found in extract from GH-stimulated cells, which indicates that this complex is preformed before and dissociates after GH stimulation of CHOA cells. In U266 cells the reverse finding has been reported (11). IFNß stimulation of these cells activated MAPK and caused MAPK to complex with STAT1a. With COS cells we showed that wild-type and serine 780 mutant of STAT5a complex with ERK1, either as a wild-type or kinase mutant form. It was only with the combination of wild-type STAT5a (containing serine 780) and wild-type ERK1 (active kinase) that less complex was seen in these GH-stimulated cells. This experiment indicates that complex formation neither requires the presence of serine 780 in full-length STAT5a nor an activated ERK. It is likely that the interacting regions of the two proteins are the C-terminal part of STAT5a, with its ERK recognition sequence, and the kinase region of ERK. It could be that other regions of the two proteins are also involved.

It has earlier been shown that the catalytic and substrate recognition domains of ERK1 are situated in the N-terminal and C-terminal parts of ERK1, respectively (25). When we used two different anti-ERK1/2 antibodies, raised against amino acids 333–367 and 339–353 of the C-terminal part of ERK, for coimmunoprecipitation of ERK and STAT5a, only weakly detectable signals of coprecipitated STAT5a were found. This was not the case with the antibody raised against full-length recombinant ERK2. One explanation for this finding could be that the C-terminal substrate recognition domain of ERK binds an unknown part of STAT5a, which results in steric hindrance of the binding site of the two different anti-ERK1/2 antibodies. One of the nuclear targets of ERK, Elk-1, which is a member of the ternary complex factor subfamily of ETS-domain transcription factors (26), has been demonstrated to bind ERK2 by a domain that is distinct from, and located N-terminally to, its phosphoacceptor motif (27). Targeting via this domain is essential for the efficient and rapid phosphorylation of Elk-1 in vitro and full rapid activation in vivo. The existence of a similar ERK-binding domain in STAT5a remains to be determined. In the experiments in which we used wild-type and serine 780 mutated C-terminal STAT5a GST-fusion constructs, GH-induced activation of ERKs resulted in increased binding of ERK1 and ERK2 to wild-type GST-fusion construct only. This indicates that activation of ERK increases the affinity of its kinase domain or enzyme active site for its recognition amino acid sequence of STAT5a in which serine 780 seems to be of key importance for successful interaction. The GST-fusion protein constructs contained the last 64 C-terminal amino acids of STAT5a. With both wild-type and serine 780-mutated GST fusion protein constructs, some background binding to ERK1 and ERK2 was seen that was higher than ERK binding to GST only. At present, it is too early to propose that this 64-amino acid long sequence contains an ERK-targeting domain. Further studies are needed to resolve this issue.

In extract from GH-treated COS cells, transfected with wild-type forms of both STAT5a and ERK1, less STAT5a-ERK1 complex was detected than in extracts from GH-treated COS cells transfected with wild-type ERK1 and serine 780-mutated STAT5a. Thus, it is possible that phosphorylation of serine 780 decreases the affinity for interaction between STAT5a and ERK1. If complex formation between STAT5a and ERK1 is due to an interaction at two sites, phosphorylation of serine 780 probably decreases the affinity for interaction at both these sites. MAPK-interacting kinase 1 (Mnk1) and ribosomal S6 kinase (RSK), both of which are activated by ERK1/2 after mitogen stimulation, have been shown to bind more strongly to inactive than active ERK, implying that they dissociate from ERK after mitogen stimulation (28, 29).

The finding of a preformed STAT5a-ERK complex before GH stimulation and its dissociation after GH stimulation makes it likely that serine phosphorylation of STAT5a occurs in cytosol. Our time course study showed that GH-induced activation of ERK1 and ERK2 and STAT5a had similar kinetics. It is not possible to conclude from this experiment which of the phosphorylations occurs first. Other experiments are needed to answer this question. Serine phosphorylation of STAT1, STAT3, and STAT5 can occur without tyrosine phosphorylation (6, 8, 30, 31). Serine phosphorylation of STAT3 has been shown to negatively modulate STAT3 tyrosine phosphorylation (9), indicating possibilites for modulation of STAT activation by serine kinases.

ERKs were initially described as microtubuli-associated proteins (19, 20). Later investigations have shown that ERKs are also present in cytosolic compartments not associated with microtubuli and also in the nucleus (21). It has been determined that one-third of the total MAPK is associated with microtubular cytoskeleton in NIH 3T3 fibroblasts, a pool that constitutes half of the detectable MAPK activity after mitogenic stimulation (20). In our present study, disruption of microtubuli with colchicine did not decrease GH-induced ERK1 and ERK2 activation and also did not inhibit STAT5a nuclear translocation. Thus, it is probable that STAT5a is not complexed with microtubuli-associated ERK. It has recently been shown that GH-induced reorganization of actin cytoskeleton is not required for STAT5-mediated transcriptional activation (32). This finding and the results presented in the present paper suggest that STAT5 nuclear transport occurs via a cytoskeleton-independent mechanism.

From our results it is not possible to estimate the amount of STAT5a complexed with ERK1 and/or ERK2. Further experiments are needed to describe the molecular events occurring after GH stimulation and the role of a preformed STAT5a-ERK1/2 complex. The prevailing dogma for activation of STATs states that, after JAK-mediated phosphorylation of tyrosine residues situated on the intracellular region of the cytokine receptor, STATs are recruited to the receptor via binding to these phosphorylated tyrosines via their SH2 domains. After receptor binding STATs are activated via JAK-mediated tyrosine phosphorylation. If preformed STAT5a-inactive ERK1/2 complexes are present in the receptor proximal environment and if tyrosine phosphorylation precedes serine phosphorylation, ERK1/2 could also be recruited to receptor upon ligand stimulation. It has been shown previously that ERK coimmunoprecipitated with IFN-{alpha}/ß receptor (11). It has been shown recently that STAT3, when bound to the IFNAR1 chain of the type I IFN receptor, itself bound phosphatidylinositol 3-kinase and thus functioned as a docking molecule (33).

Our in vitro kinase experiments with C-terminal STAT5a-GST fusion protein and recombinant full-length STAT5a showed that serine 780 is the only target for ERK under these conditions. This does not exclude that serine 780 can be phosphorylated by other serine kinases. Epidermal growth factor or interleukin-6 treatment of the same cell line has been reported to induce phosphorylation of serine 727 in STAT3 by ERK-dependent or ERK-independent pathways, respectively (9). This shows that different hormones, acting on the same cell, can induce serine phosphorylation through activation of different serine kinases. Furthermore, the same hormone can activate different pathways in different cells to induce serine phosphorylation of STAT3 (34, 35). It is possible that serine phosphorylation of STAT5a can also occur at other sites than serine 780. Recently, Yamashita et al. (36) identified serine 725 of STAT5a, within a PSP motif, as being constitutively phosphorylated in COS and Nb2 cells. Their data also suggested the existence of a second major serine phosphorylation site in STAT5a, which was not identified. The constitutive phosphorylation of STAT5a was shown to be suppressed by the MEK inhibitor PD98059. Interestingly, in STAT5b, which is approximately 90% homologous to STAT5a but lacks the C-terminal ERK recognition sequence (14, 37), phosphorylation of the corresponding serine (730) was stimulated by PRL but was not suppressed by PD98059. Mutational analysis showed that phosphorylation of serine 725 in STAT5a and serine 730 in STAT5b was not essential for DNA binding and transcriptional activation. Other studies have shown that STAT5a and b are both serine phosphorylated by ERK-independent pathways, constitutively in a mouse mammary epithelial cell line (38) and after interleukin-2 treatment of T lymphocytes (39, 40).

In our earlier study we showed that inhibition of MEK resulted in abolished GH-induced ERK activation and decreased capacity of STAT5a to activate transcription. From these data we concluded that ERK was involved. It cannot be totally excluded that another mechanism was responsible for the effect of MEK inhibition. Transfection experiments with COS cells, in the present study, showed that the presence of the inactive form of ERK1 decreased the capacity of STAT5a to activate reporter gene transcription. Thus, this experiment confirms the involvement of ERK in regulation of STAT5a functional capacity.

In our earlier study we proposed that ERK phosphorylates serine 780 in STAT5a. In the present study experiments were performed that lend further support to this notion. From results described in our present work the following model for interaction between ERK and STAT5a in CHOA cells can be proposed (Fig. 7Go). In unstimulated cells STAT5a is complexed with inactive ERK. ERK binds to STAT5a via its C-terminal substrate recognition domain to an unknown region on STAT5a and via its active site to the C-terminal ERK recognition sequence in STAT5a. Upon GH stimulation, MEK activates ERK through phosphorylation of specific threonine and tyrosine residues in ERK. Active ERK phosphorylates serine 780 in STAT5a, resulting in decreased affinity between the two proteins and dissociation of the complex.



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Figure 7. Proposed Model for STAT5a Interaction with ERK in CHOA Cells

In unstimulated cells STAT5a is complexed with inactive ERK. ERK binds to STAT5a via its C-terminal substrate recognition domain to an unknown region on STAT5a and via its active site to the C-terminal ERK recognition sequence in STAT5a. Upon GH stimulation, MEK activates ERK through phosphorylation of specific threonine and tyrosine residues in ERK. Active ERK phosphorylates serine 780 in STAT5a, resulting in decreased affinity between the two proteins and dissociation of the complex.

 
Several STAT factors have now been described to be serine phosphorylated (2). In some cases, ERK has been shown to be the kinase involved (9, 11). More studies are needed to identify other kinases taking part in serine phosphorylation of STAT factors as well as additional sites on STATs that can be serine/threonine phosphorylated. The biological importance of cross-talk between ERK and STAT5a is at present unknown. In our present study, MAPK pathway was activated by GH, simultaneously with activation of JAK2/STAT5 pathway. As MAPK pathway is also activated by several other hormones and growth factors, this could be a means for these factors to modulate GH-regulated gene expression. This remains to be determined. The consequence of serine phosphorylation of STAT5a C-terminal transactivation domain is at present also unknown. In our earlier study we found that inhibition of serine phosphorylation did not inhibit nuclear translocation of STAT5a but only decreased the amount of STAT5 in nuclei with DNA-binding capacity (13). We suggested that this could be due to increased phosphotyrosine dephosphorylation of non-serine-phosphorylated STAT5a. This does not exclude that other mechanisms, such as STAT5a dimer stability, its interaction with proteins belonging to the transcriptional machinery, or proteasome-dependent breakdown of STAT5a, are influenced by serine phosphorylation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
CHO cells, stably transfected with rat GH receptor cDNA (CHOA), were grown to 50% confluence in 100-mm culture dishes using Ham’s F-12 medium (Life Technologies, Gaithersburg, MD) containing 10% FCS, 50 U/ml penicillin, and 50 µg/ml streptomycin. After washing with PBS, cells were incubated in serum-free medium for approximately 16 h before treatment with human GH (hGH), a generous gift from Pharmacia & Upjohn, Stockholm, Sweden. For mictrotubular disruption, cells were pretreated with 10 µM colchicine (Sigma Chemical Co., St. Louis, MO) 60 min before GH treatment.

Preparation of Cellular Extracts
After treatment with hGH for indicated times, cultured cells were rinsed with ice-cold PBS and frozen on dry ice. The cells were then scraped into an RSB buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 6 mM MgCl2, 1 mM dithiothreitol, 0.1 mM Na3VO4) and disrupted with a Dounce homogenizer. The supernatant obtained after centrifugation was used as cytosolic extract. The nuclear pellet obtained after centrifugation was resuspended in 3 volumes of extraction buffer (20% glycerol, 20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM dithio-threithol, 0.1 mM Na3VO4) and incubated on ice for 30 min. The supernatant obtained after centrifugation was used as nuclear extract. Protein concentration was measured by the Bradford method.

Western Blotting
Western blotting was performed as previously described (13). The membranes were incubated with primary polyclonal antibodies, anti-ERK1/2 [dilution, 1:500; StressGen (Biotechnologies Corp., Victoria, Canada) and Transduction Laboratories (Lexington, KY)], anti-STAT5a (dilution, 1:750; Santa Cruz Biotechnology, Santa Cruz, CA) and antiactive ERK (dilution, 1:20000; Promega, Madison, WI). The membranes were analyzed with the enhanced chemiluminescence method (Amersham, Arlington Heights, IL).

GST-Fusion Proteins
The expression plasmids for wild-type STAT5a, pME18S-STAT5a, and serine 780-mutated STAT5a, pME18S-STAT5aS780A (13), were used to PCR amplify the C-terminal end of STAT5a and STAT5aS780A, including the codons for the last 64 amino acids (aa 730-aa 794), using the synthetic oligonucleotides, 5'-TCGAGAATTCCCCTCAACCTCACTACAACATG-3' (containing EcoRI cloning site) and 5'-TCAGCTCGAGTCAGGACAGGGAGCTTCTAGC-3' (containing XhoI cloning site). The oligos were synthesized by Cybergene (Huddinge, Sweden). The PCR products were ligated into the EcoRI/XhoI sites of the GST fusion vector, pGEX-4T3 (Pharmacia Biotech). Expression and purification of GST-fusion proteins were done according to the manufacturer’s instructions (Pharmacia Biotech). GST-fusion proteins were used in coprecipitation and in vitro kinase experiments.

Coprecipitation with GST-Fusion Protein
Four micrograms of GST-fusion proteins were incubated for 45 min at room temperature with cytosolic extract from CHOA cells, treated with or without 100 nM GH for 10 min, in a HEPES buffer (20 mM HEPES, pH 7.6, 1 mM EDTA, 1 mM dithiothreitol, 1 mM Na3VO4, 10 mM NaF, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 2 µg/ml pepstatin A). A 40-µl slurry (50:50) of glutathione-Sepharose (Pharmacia Biotech) was added to incubations, and after 30 min the precipitations were washed three times with PBS containing 0.1% Triton X-100. The precipitation were boiled in SDS solubilization buffer after which samples were separated by SDS-PAGE and analyzed by Western blotting technique with a polyclonal antibody against ERK1/2.

Baculo Virus-Expressed Proteins
The plasmids pME18S-STAT5a and pME18S-STAT5aS780A were PCR amplified using the synthetic oligonucleotides, 5'-ATCGGAATTCAAATGGCGGGCTGGAT-TCAG-3' (containing the EcoRI cloning site) and 5'-TCAGTCTAGATCAGGACAGG-GAGCTTCTA-3' (containing the XbaI cloning site). The oligos were synthesized by Cybergene (Huddinge, Sweden). The PCR products were ligated into the EcoRI/XbaI cloning site of the pFastBac HTB vector (Bac-to-Bac baculovirus expression system, Life Technologies). Clones were identified and both strands were sequenced by automatic sequencing. Further steps, including transformation of the plasmids into DH10 Bac cells containing bacmid and helper DNA, isolation of recombinant bacmid DNA, transfection into Sf9-insect cells, and protein expression, were done according to the manufacturer’s instruction (Life Technologies). His-tagged recombinant proteins were purified with nickel resin and used in coprecipitation and in vitro kinase experiments.

In Vitro Phosphorylation
Purified GST-fusion proteins or recombinant wild-type or mutated STAT5a were incubated in a kinase buffer (Promega), 1 µCi [{gamma}-32P]ATP (Amersham)]/sample and 50 ng active ERK (Promega) in a final incubation volume of 50 µl. The incubation was allowed to proceed for 20 min at 30 C, after which the reaction was terminated with SDS-solubilization buffer, run on an SDS-gel, and visualized by autoradiography and PhosphorImager (Fuji Photo Film Co., Ltd, Japan).

Immunoprecipitation
Cellular extracts were diluted to 500 µl with TMB-buffer (50 mM Tris, pH 7.4, 10 mM MgCl2, 0.1% BSA, 1 mM Na3VO4, 10 mM NaF, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin and 2 µg/ml pepstatin A) and incubated with either 5 µg polyclonal anti-ERK1/2 (Stress Gene and Transduction Laboratories), 5 µg polyclonal anti-ERK2 (ERK2 immunoprecipitation antibody; Upstate Biotechnology, Inc., Lake Placid, NY) or 5 µg monoclonal phosphotyrosine antibody (PY20; Transduction Laboratories) for 45 min at room temperature. After incubation for 30 min with 30 µl (50:50 slurry) of protein A-Sepharose (Pharmacia Biotech), immune complexes were washed three times with TMB buffer and further processed for Western blot analysis as previously described (13).

Gel Electrophoresis Mobility Shift Assay
Gel electrophoresis mobility shift assay was performed according to standard protocol previously described (24). An oligonucleotide containing the STAT5 binding, {gamma}-interferon activated sequence (GAS)-like element from the ß-casein promoter (TGCTTCTTGGAATT) was used as a probe (18). Competition studies were performed with the following double-stranded oligonucleotides representing various GAS-like promoter elements from cytokine-regulated genes (41): human guanylate binding protein (GBP)-ATTACTCTAAA; human FC{gamma} receptor type 1 (Fc{gamma}R1)-TTTCCCAGAAA; human IFN consensus sequence binding protein (ICSBP)-TTTCTCCGAAA; human IFN-induced protein 53 (IFP-53)-ATTCTCAGAAA; human IFN-regulatory factor 1 (IRF-1)-TTTCCCCGAAA; mouse Ly 6E antigen (Ly6E)-ATTCCTGTAAG; and mouse monokine induced by IFN-{gamma} (Mig)-CTTACTATAAA.

Cellular Transfection and Luciferase Assay
Transfection of COS cells and luciferase assay were performed as previously described (13). In short, 50% confluent 30-mm dishes with COS cells were transfected with 1 µg of STAT5-regulated reporter plasmid pSPI-GLE1-luc (12) and expression plasmids for STAT5a (0.2 µg; pME18S-STAT5a), GHR (0.2 µg; pLM108), and wt-ERK1 (0.3 µg; pcDNA1/Neo-HA-ERK1-wt) or the kinase inactive ERK1 (0.3 µg; T192A, pcDNA1/Neo-HA-ERK1-kin-) or control plasmid (0.3 µg; pcDNA) and ß-gal (0.05 µg; pCMV-gal). Luciferase and ß-galactosidase activities were measured in 96-well plates in the luminometer Arthos Lucy1 (Arthos Labtec Instruments, Salzburg, Austria) after automatic injection of reagent.

For precipitation studies 50% confluent 100-mm dishes with COS cells were transfected with the expression vectors (1 µg of each plasmid) for the GH receptor and STAT5a or STAT5aS780A and HA-tagged wild-type ERK1 (HA-ERK-wt) or HA-tagged kinase inactive ERK1 (HA-ERK-kin-) and pcDNA. Transfections were performed with DOTAP (Boheringer Mannheim, Mannheim, Germany). The last 16 h before harvest the COS cells were grown in serum-free DMEM, after which the cells were treated with GH for 8 min, and the cytosolic extract was prepared as described.


    ACKNOWLEDGMENTS
 
We thank Dr. Alice F. Mui for STAT5a expression vector, Dr. Jacques Pouysségur and Dr. Pär Gerwins for ERK1-wild-type and ERK1-kinase-inactive expression plasmids, and Dr. Tim Wood for SPI-GLE1-luciferase reporter gene.


    FOOTNOTES
 
Address requests for reprints to: Dr. Lars-Arne Haldosén, Department of Medical Nutrition, Karolinska Institute, Novum, S-141 86 Huddinge, Sweden. E-mail: Lars-Arne.Haldosen{at}mednut.ki.se

This work was supported by grants from the Swedish Cancer Society (Grant 3020-B97–04XBB), the Swedish Medical Research Council (Grant 03X-06807), Karolinska Institutet, and Magnus Bergvalls Stiftelse.

Received for publication May 28, 1998. Revision received December 10, 1998. Accepted for publication January 8, 1999.


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