Association of 2',5'-Oligoadenylate Synthetase with the Prolactin (PRL) Receptor: Alteration in PRL-Inducible Stat1 (Signal Transducer and Activator of Transcription 1) Signaling to the IRF-1 (Interferon-Regulatory Factor 1) Promoter

Kathleen M. McAveney1,2, Melissa L. Book2, Pin Ling, Judith Chebath and Li-yuan Yu-Lee

Departments of Medicine (K.M.M., L-y.Y.-L.), Molecular and Cellular Biology (M.L.B., L.-y.Y.-L), and Immunology (P.L., L.-y.Y.-L.) Baylor College of Medicine Houston, Texas 77030-3411
Department of Molecular Genetics (J.C.) Weizmann Institute of Sciences Rehovot, Israel 76100


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The PRL receptor (PRL-R) signals through the Janus tyrosine kinases (JAK) and other non-JAK tyrosine kinases, some of which are preassociated with the PRL-R. To clone PRL-R interacting proteins, the intracellular domain (ICD) of the long form of the PRL-R was used in a yeast two-hybrid screen of a human B cell cDNA library. One PRL-R interacting protein was identified as the 42-kDa form of the enzyme 2',5'-oligoadenylate synthetase (OAS). The in vivo interactions in yeast were further confirmed by an in vitro interaction assay and by coimmunoprecipitation in transfected mammalian cells. Functionally, OAS reduced the basal activity of two types of promoters in transiently transfected COS-1 cells. In the presence of PRL, OAS inhibited PRL induction of the immediate early IRF-1 (interferon-regulatory factor 1) promoter, but not PRL induction of the differentiation-specific ß-casein promoter, suggesting that OAS exerts specific effects on immediate early gene promoters. The inhibitory effects of OAS were accompanied by a reduction in PRL-inducible Stat1 (signal transducer and activator of transcription 1) DNA binding activity at the IRF-1 GAS (interferon-{gamma}-activated sequence) element. These results demonstrate a novel interaction of OAS with the PRL-R and suggest a role for OAS in modulating Stat1-mediated signaling to an immediate early gene promoter. Although previously characterized as a regulator of ribonuclease (RNase) L antiviral responses, OAS may have additional effects on cytokine receptor signal transduction pathways.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The PRL receptor (PRL-R) is a member of the hematopoietin/cytokine receptor superfamily whose members are involved in mediating the growth and differentiation of both hematopoietic and nonhematopoietic cells (1, 2). PRL-R is ubiquitously expressed in lymphocytes (3, 4), and the PRL-R (5, 6) as well as PRL (7) have been cloned from T lymphocytes. These findings support a role of PRL as a hematopoietic cytokine (2). Three forms of the PRL-R have been cloned: long, Nb2, and short, which result from alternative splicing of 3'-end exons from a single gene (8). In addition, the Nb2 PRL-R contains an in-frame deletion of 198 amino acids, which generates a shortened form relative to the long PRL-R intracellular domain (ICD) (9).

The PRL-R signals through the JAK (Janus kinase)/Stat pathway (10), which was first described for the interferon (IFN) signaling pathway (11). In the case of the PRL-R, monomeric receptors dimerize upon ligand binding, which is followed by the activation of the receptor-associated protein tyrosine kinase JAK2 (12), and the subsequent phosphorylation of critical tyrosine residues in the PRL-R (13, 14). JAK2 is also responsible for phosphorylating tyrosine residues in a family of latent cytoplasmic transcription factors, collectively called signal transducers and activators of transcription (Stat) (10). Upon activation, Stat proteins form homodimeric or heteromeric complexes, translocate into the nucleus, bind to conserved DNA elements in the promoters of various genes, and regulate gene transcription. Stat1, Stat3, Stat5a, and Stat5b are activated by PRL stimulation (15, 16, 17, 18). Stat1 has been shown to bind to the IFN{gamma}-activated sequence (GAS) (14) and is a positive mediator of PRL signaling to the immediate early gene, interferon regulatory factor-1 (IRF-1) (19, 20). Surprisingly, Stat5a and Stat5b both inhibit PRL signaling to the IRF-1 promoter (21). The inhibitory action of Stat5 at the IRF-1 promoter is in contrast to its stimulatory activities at other PRL-inducible differentiation-specific promoters, including ß-casein (17), ß-lactoglobulin (22), whey acidic protein (23), and {alpha}2-macroglobulin (1577).

Other molecules that have been implicated in PRL signaling include protein tyrosine phosphatases (24), as well as Vav (25), Cbl (26), Fyn (27), Raf (28), Ras (29, 30, 31), Src (32), protein kinase C (33), p38 mitogen-activated protein kinase (34), JNK (35), and ZAP70 (36). It remains to be determined how the activities of these molecules affect PRL-inducible proliferative, differentiative, and antiapoptotic responses in various target tissues. JAK2, Fyn, and Raf have been shown to be preassociated with the inactive monomeric PRL-R (27, 28, 37, 38, 39). Because prebound receptor-associated proteins are potential signaling molecules, it is feasible to use genetic means such as the yeast two-hybrid system (40, 41, 42) to identify some of these proteins by protein-protein interactions.

To further investigate the PRL signaling pathway, we used the ICD as bait to clone PRL-R interacting proteins (42). From this screen, we obtained a clone (21/8) that displayed 100% identity with the 42-kDa form of the enzyme 2',5'-oligoadenylate synthetase (OAS), a metabolic enzyme originally identified as a regulator of the ribonuclease L (RNase L) pathway during viral infection (43, 44, 45). The in vivo interaction in yeast was further confirmed by an in vitro interaction assay and by coimmunoprecipitation from mammalian cells. The functional significance of the PRL-R/OAS interaction in receptor signaling was examined by transient cotransfection with the PRL-R and several reporter CAT (chloramphenicol acetyltransferase) constructs into COS cells. Our studies suggest that OAS may be exerting a specific inhibitory effect on signaling to an immediate early gene promoter, IRF-1, but not to the differentiation-specific ß-casein promoter. Further, OAS appears to be exerting this inhibitory effect at the IRF-1 promoter by reducing PRL-inducible Stat1 DNA binding at the IRF-1 GAS element, in a dose-dependent manner. In contrast, PRL-stimulated Stat5b binding to the IRF-1 GAS element is unaltered by the presence of OAS, in agreement with the lack of OAS inhibition of the ß-casein promoter. Together, these studies suggest a novel role for the enzyme OAS in PRL-R signaling and elucidate a novel function of OAS in the transcriptional regulation of an immediate early gene through its effects on Stat1.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PRL-R Interacting Proteins
To identify novel proteins associated with the PRL-R, the yeast two-hybrid system was used to screen a human B lymphoblastoid cell cDNA expression library (42). The 50-kDa ICD of the long form of the rat PRL-R was used as a bait to clone interacting proteins. Protein-protein interaction in yeast was scored by the ability to grow on the selection medium supplemented with 50 mM 3-aminotriazole. Forty PRL-R ICD-interacting clones were isolated, seven of which were sequenced. Most of the clones either exhibited no known identity in GenBank or show some identity with expressed sequence tag (EST) clones identified by random sequencing of the human genome (data not shown). One clone (21/8) encodes the full-length 42-kDa form of the enzyme OAS (46, 47). Yeast transformed with both PRL-R ICD and OAS constructs produced blue color in the ß-galactosidase assays within 2 h, indicating efficient interaction (data not shown). These results indicate a strong protein-protein interaction between PRL-R ICD and OAS, and thus the functional significance of their interaction is the focus of this study.

OAS and PRL-R Interact in Vitro and in Vivo
To confirm the in vivo interaction in yeast, an independent in vitro interaction assay was performed. The long PRL-R ICD was cloned into the maltose binding protein (MBP) bacterial expression system to generate an MBP-PRL-R ICD (MBP-ICD) fusion protein. Either MBP control or MBP-ICD fusion protein was incubated with [35S]methionine-labeled OAS, which was generated in an in vitro transcription/translation system (21). Bound proteins were resolved on SDS-PAGE and analyzed by autoradiography (Fig. 1Go). Although 2-fold more MBP than the MBP-ICD fusion protein was used, as seen in the Coomassie stain of the gel (Fig. 1AGo), the MBP-ICD fusion protein matrix reproducibly retained substantially more of the [35S]methionine-labeled OAS than the MBP control (Fig. 1BGo). To determine whether OAS interacts with the PRL-R in mammalian cells, Flag-tagged OAS (48) was cotransfected with the long form of the PRL-R into COS-1 cells and immunoprecipitated (IP) with anti-Flag antibodies coupled to beads. Proteins retained by this Flag-tagged OAS matrix were then immunoblotted (IB) with anti-PRL-R antibodies (Fig. 1CGo). The long form of the PRL-R (80 kDa) was retained by the Flag-tagged OAS matrix (Fig. 1CGo, lane 2), demonstrating that the PRL-R can interact with the 42-kDa OAS in mammalian cells. Some degradation of the overexpressed PRL-R was observed, along with a nonspecific band which was also evident in the empty vector-transfected control cells (Fig. 1CGo, lane 1). Interestingly, the Nb2 (65-kDa) form of the PRL-R, which interacts with OAS in the yeast two-hybrid interaction assay (data not shown), also coimmunoprecipitated with Flag-tagged OAS (Fig. 1CGo, lane 3). Equal expression of Flag-OAS was verified by immunoblotting with anti-OAS antibodies (data not shown). Thus, the 42-kDa form of the OAS interacts with the PRL-R in vitro and in vivo.



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Figure 1. PRL-R Interacts with OAS in an in Vitro Interaction Assay and in Mammalian Cells

MBP control or MPB-PRL-R ICD fusion proteins were incubated for 1 h at 4 C with in vitro-translated [35S]methionine-labeled OAS as described in Materials and Methods. The MBP matrix proteins (A) and the bound proteins (B) were resolved by 10% SDS-PAGE and stained with Coomassie blue (A), and the dried gel was analyzed by autoradiography for radiolabeled OAS (B). The data shown are representative of three independent experiments. C, Coimmunoprecipitation (Co-IP). COS-1 cells were cotransfected with Flag-tagged OAS and either empty vector control (lane 1), the long form of the PRL-R (lane 2), or the Nb2 form of the PRL-R (lane 3). Cell lysates were immunoprecipitated (IP) with anti-Flag antibodies coupled to beads, and the precipitated proteins were then immunoblotted (IB) with anti-PRL-R antibodies that recognize the ICD of both the long and Nb2 forms of the PRL-R. The data are representative of two independent experiments.

 
OAS Inhibits PRL Signaling to the Immediate Early Gene IRF-1
Having established a physical association between OAS and the PRL-R, the functional significance of OAS in PRL-R signaling was next examined. A series of transient cotransfection experiments was employed. COS-1 cells were transfected with constructs containing OAS or vector control, the long form of the PRL-R, and either of two well described PRL-responsive promoter reporters. One is the immediate early gene IRF-1 promoter (1.7-kb IRF-1-CAT) (14, 49), and the other is the differentiation-specific milk protein ß-casein promoter (2.3-kb ß-casein-CAT) (21, 50) (Fig. 2Go). The transfections also included Stat1 or Stat5b to ensure robust PRL-inducible IRF-1 or ß-casein promoter activities, respectively, as previously described (14, 21). Overexpression of OAS led to a 40–60% decrease in basal activity from both the IRF-1 (Fig. 2AGo) and ß-casein (Fig. 2BGo) promoters. The decrease in basal promoter activity is not likely due to a general effect of OAS on cellular RNA metabolism via activation of the RNase L pathway (45), as a similar OAS-related reduction in basal IRF-1 promoter activity was also observed in RNase L-/- mouse embryo fibroblasts (data not shown). Although the exact mechanism of OAS-mediated decrease in basal promoter activity is unclear, our interpretation is that OAS is affecting some component of the basal transcriptional machinery used by both the ß-casein and the IRF-1 promoters.



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Figure 2. OAS Inhibits Signaling to the Immediate Early IRF-1 Promoter but Not the Differentiation-Specific ß-Casein Promoter

COS-1 cells were transiently transfected with 1 µg each of constructs expressing the long form (A and B) or the Nb2 form (C and D) of the PRL-R, OAS (solid bars), or vector control (open bars), and 1.7 kb IRF-1-CAT (0.2 µg) (A and C) plus Stat1 or 2.3 kb ß-casein-CAT (1 µg) (B and D) plus Stat5b. Cells were made quiescent overnight in 1% horse serum medium and then stimulated with 100 ng/ml PRL for 24 h. Cell lysates were assayed for CAT enzyme activity, which was expressed as counts per min of 14C-chloramphenicol converted/µg protein assayed. The overall lower activity of the ß-casein promoter in COS cells has been previously observed (21 ). Data are representative of four (A and B) or two (C and D) independent experiments, with each condition assayed in triplicate. Error bars indicate SEM.

 
In the presence of PRL, overall IRF-1 promoter activity was also decreased approximately 60% in the presence of exogenous OAS, to the level of the uninduced vector controls (Fig. 2AGo). This inhibition of PRL signaling to the IRF-1 promoter is specific, as OAS did not alter PRL signaling to the ß-casein promoter (Fig. 2BGo). This observation suggests that OAS does not alter the stability of the chloramphenicol acetyltransferase (CAT) mRNA, but that OAS may be affecting specific signaling to the IRF-1 promoter. Additionally, this OAS-mediated inhibition of PRL signaling to the IRF-1 promoter was observed with the Nb2 form of the PRL-R (Fig. 2CGo). As observed with the long form of the PRL-R, OAS also did not alter PRL signaling through the Nb2 PRL-R to the ß-casein promoter (Fig. 2DGo). These results indicate that OAS is exerting a specific effect on PRL signaling to an immediate early gene promoter, and this effect is observed with both the long and the Nb2 forms of the PRL-R.

Previous studies have shown that Stat1 is a major mediator of PRL signaling to the IRF-1 promoter (14) while Stat5 mediates PRL signaling to the ß-casein promoter (17, 21). We next determined whether OAS might be exerting a specific effect on Stat1-mediated signaling to the IRF-1 promoter. To further examine the effect of OAS on Stat1 function, the 0.2-kb IRF-1 promoter, which contains a single PRL-inducible and Stat1-responsive GAS element (20, 49), and a heterologous thymidine kinase (TK) promoter containing three copies of the IRF-1 GAS element (3C GAS-TK-CAT) (14), were examined by transient transfection into COS cells (Fig. 3Go). As compared with the 1.7-kb IRF-1 promoter (Fig. 3AGo), OAS similarly inhibited both basal and PRL-inducible activity at the 0.2-kb IRF-1 promoter (Fig. 3BGo), suggesting an effect of OAS on Stat1 signaling to the 0.2-kb IRF-1 promoter. Further, OAS reduced PRL activation of the 3C GAS-TK promoter (Fig. 3CGo), thus verifying its ability to inhibit Stat1-mediated signaling to the IRF-1 GAS. Together, these results suggest that OAS specifically reduces PRL-inducible Stat1-mediated signaling to the IRF-1 promoter.



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Figure 3. OAS Inhibits Stat1-Mediated IRF-1 Promoter Activity

COS-1 cells were transiently transfected with 1 µg each of the long form of the PRL-R, OAS (solid bars), or vector control (open bars), Stat1 and (A) the 1.7 kb IRF-1-CAT (0.2 µg) (n = 2) (A), 0.2 kb IRF-1-CAT (0.2 µg) (n = 4) (B), or 3C GAS TK-CAT (0.5 µg) (n = 3) (C). Cells were treated with PRL and assayed in triplicate as described in Fig. 2Go. Data for each construct were normalized against the +PRL vector controls (set at 100% maximum) within each experiment, and two to four independent experiments are represented. Error bars indicate SEM.

 
OAS Reduces Stat1 DNA Binding to the IRF-1 GAS Element
To determine whether OAS inhibits PRL stimulation of the IRF-1 promoter through a specific effect on Stat1 DNA binding activity, we examined PRL-inducible Stat1 binding to the IRF-1 GAS element in an electrophoretic mobility shift assay (EMSA) (Fig. 4Go). Whole-cell extracts were prepared from COS cells transiently cotransfected with the long form of the PRL-R, Stat1, and either OAS or empty vector controls. PRL enhanced the formation of a complex that binds to the IRF-1 GAS element (Fig. 4AGo, lanes 2 and 3). This binding is specific as it is competed by 50x molar excess of cold IRF-1 GAS oligo (Fig. 4AGo, lane 4). This PRL-inducible complex contains Stat1 as it can be supershifted by anti-Stat1 antibodies (SS 1) (Fig. 4AGo, lane 5) but not by anti-Stat6 antibodies (Fig. 4AGo, lane 6). A band comigrating with the Stat1 complex was observed in control cells (Fig. 4AGo, lane 1). The nature of this complex is unclear, but it does not contain Stat1, as it is neither supershifted by anti-Stat1 antibodies (Fig. 4AGo, lane 5) nor diminished by the presence of exogenous OAS (Fig. 4AGo, lane 7). Interestingly, in the presence of exogenous OAS, PRL-inducible Stat1 binding to the IRF-1 GAS element was markedly and reproducibly reduced by 40% (Fig. 4AGo, lane 8). This decrease in Stat1 binding to the IRF-1 GAS element correlates with the decrease in PRL-inducible Stat1-mediated IRF-1 promoter activity in the presence of exogenous OAS (Fig. 2Go, A and C, and Fig. 3Go), as measured under identical transfection conditions employing 1 µg each of OAS and Stat1. Again, the specificity of the OAS-inhibited PRL-inducible complex was shown by competition with excess cold IRF-1 GAS oligo (Fig. 4AGo, lane 9) and by a supershift with anti-Stat1 (Fig. 4AGo, lane 10) but not anti-Stat6 antibodies (Fig. 4AGo, lane 11).



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Figure 4. OAS Modulates PRL-Inducible Stat1 DNA Binding

A, OAS reduces Stat1 DNA binding activity at the IRF-1 GAS. Whole-cell extracts were prepared from 30-min PRL-stimulated COS-1 cells transiently transfected with 1 µg each of the long form of the PRL-R, Stat1, and OAS (lanes 7–11) or vector control (lanes 2–6), and used for EMSA with a radiolabeled IRF-1 GAS element as a probe. The specificity of the protein/DNA complex was determined by competition with 50x molar excess of cold probe (lanes 4 and 9) and by incubation with anti-Stat1 monoclonal antibodies (lanes 5 and 10) or isotype-matched control anti-Stat6 monoclonal antibodies (lanes 6 and 11). B, OAS does not affect Stat5b DNA binding activity at the IRF-1 GAS. Whole cell lysates from COS-1 cells transfected as in panel A were used except that Stat5b was employed with either OAS (lanes 4 and 5) or vector control (lanes 2 and 3 and 6–9), and examined by EMSA with a radiolabeled IRF-1 GAS probe (lane 1). The specificity of the protein/DNA complex was determined by competition with 50x molar excess of cold probe (lane 6) and by incubation with anti-Stat5b antibodies (21 ) (lane 7), preimmune serum (lane 8), or anti-Stat1 antibodies (lane 9). C, OAS does not alter the kinetics of Stat1 binding to the IRF-1 GAS. COS-1 cells were transfected as in panel A and stimulated with PRL from 0 to 4 h as indicated. Whole-cell lysates were examined by EMSA using an IRF-1 GAS probe (lane 1). Lanes 2–5, vector-transfected control cells; lanes 6–9, OAS-transfected cells. D, OAS reduces Stat1 DNA binding activity in a dose-dependent manner. COS-1 cells were transfected as in panel A except that increasing concentrations of OAS as indicated were employed. Lanes 2 and 3, No exogenous OAS; lanes 4 and 5, 0.1 µg OAS; lanes 6 and 7, 1 µg OAS; lanes 8 and 9, 2 µg OAS. Each panel (A–D) is a representative EMSA from three to five experiments using extracts from two independent transfections. Stat1, Stat1/GAS complex; Stat5, Stat5/GAS complex; SS 1, supershifted Stat1 complex; SS 5, supershifted Stat5 complex; NS, nonspecific binding. E, OAS does not reduce Stat1 protein levels. COS-1 cells were transfected and stimulated with PRL as indicated. Whole-cell lysates were subjected to SDS-PAGE, transferred to Immobilon P membrane, and immunoblotted with polyclonal anti-Stat1 antibodies. Lane 1, no exogenous OAS; lanes 2 and 3, 0.1 µg OAS; lanes 4 and 5, 1 µg OAS; lanes 6 and 7, 2 µg OAS. A representative blot from three independent transfection experiments is shown.

 
As shown in Fig. 2Go, B and D, PRL signaling through Stat5b to the ß-casein promoter was not affected by the presence of OAS. To investigate the effects of OAS on Stat5b binding to the IRF-1 GAS, COS cells were transiently cotransfected with the long form of the PRL-R, Stat5b, and OAS or vector control. PRL stimulated a complex that binds to the IRF-1 GAS (Fig. 4BGo, lanes 2 and 3), as shown previously (21). The specificity of this binding was verified by 50x molar excess competition with cold GAS oligos (Fig. 4BGo, lane 6). The complex contains Stat5b, as it can be supershifted by anti-Stat5b antibodies (SS 5) (21) (Fig. 4BGo, lane 7) but not by preimmune serum (Fig. 4BGo, lane 8) or anti-Stat1 antibodies (Fig. 4BGo, lane 9). The dark complex migrating near the top of the gel is due to nonspecific (NS) interaction with serum proteins as it was also observed with preimmune serum (Fig. 4bGo, lanes 7 and 8). Interestingly, the presence of exogenous OAS did not affect PRL-inducible Stat5b binding to the IRF-1 GAS element (Fig. 4BGo, compare lane 3 to lane 5). Indeed, OAS seems to reproducibly enhance Stat5b binding to the IRF-1 GAS, the basis of which is not understood. The nature of the slower-migrating complex above the Stat5-containing complex is currently unclear. These results show that OAS does not affect Stat5b binding to DNA and agree with the functional data that show that OAS has no effect on PRL-inducible Stat5b-mediated signaling to the differentiation specific ß-casein promoter (Fig. 2Go, B and D).

To further characterize the changes in Stat1 interaction with the IRF-1 GAS in the presence of exogenous OAS, we investigated Stat1 binding across a PRL induction time course. As before, COS cells were transiently cotransfected with the long form of the PRL-R, Stat1, and OAS and were stimulated with PRL for the times indicated (Fig. 4CGo). Stat1 binding to the IRF-1 GAS was detected within 30 min of PRL stimulation and persisted for 4 h post stimulation (Fig. 4CGo, lanes 3–5). Although the presence of exogenous OAS decreased the level of Stat1 binding, OAS did not alter the kinetics of Stat1 binding to the IRF-1 GAS element (Fig. 4CGo, lanes 7–9). Next, we asked whether the reduction in Stat1 binding was dependent on the amount of OAS transfected. COS cells were transiently cotransfected with the long form of the PRL-R, Stat1, and increasing amounts of OAS (0, 0.1, 1.0, and 2.0 µg), and extracts were analyzed by EMSA (Fig. 4DGo). PRL stimulated Stat1 binding to the IRF-1 GAS element (Fig. 4DGo, lanes 2 and 3). Increasing concentrations of exogenous OAS led to a dose-dependent decrease in the level of both basal as well as PRL-inducible Stat1 binding to the IRF-1 GAS element (Fig. 4DGo, lanes 4–9). Interestingly, total Stat1 protein levels (Fig. 4EGo) and Stat1 mRNA levels (data not shown) do not change in the presence of increasing amounts of transfected OAS. These results show that OAS mediates a decrease in PRL-inducible Stat1 binding to the IRF-1 GAS element in a dose-dependent manner, in the absence of any change in Stat1 protein levels.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PRL is a hormone/cytokine that induces target cells to proliferate and/or differentiate, depending upon cell lineage and stage of differentiation. Using the long form of the PRL-R intracellular domain as bait, we cloned the 42-kDa metabolic enzyme OAS as a PRL-R interacting protein and demonstrated a novel role for OAS in the PRL-R signal transduction pathway. Functionally, OAS specifically inhibits the activation of an immediate early gene IRF-1 that is normally activated in response to PRL stimulation of both T cells (20) and normal mouse (51) and rat leukocytes (Z. Dogusan, M.L. Book, P. Verdood, L.-y. Yu-Lee, and E. Hooghe-Peters, submitted). Stat1 binding to the GAS element has been shown to be critical in mediating PRL activation of the IRF-1 promoter (14, 20). Exogenous OAS specifically inhibits IRF-1 promoter activity by reducing PRL-inducible Stat1 binding to the IRF-1 GAS element without reducing Stat1 protein levels. The ability of OAS to regulate IRF-1 promoter activity indicates an intriguing and novel role of OAS in cytokine receptor-mediated signal transduction.

The 2',5'-oligoadenylate synthetases (OAS) are a family of widely expressed enzymes that catalyze the synthesis of 2',5'-linked oligomers of adenosine (2, 3, 4, 5) from ATP in response to activation by IFN or double-stranded RNA (53). The 2–5A oligomers bind to and activate RNase L which regulates RNA metabolism and affects protein synthesis (45). Three major forms of human OAS that exhibit distinct patterns of cellular localization and sensitivity to IFN activation have been identified: p42/p46, p69/p71, and p100 (47, 54). The 42-kDa OAS isoform is found in the cytosol by subcellular fractionation (55). All of the OAS proteins bind ATP, are activated by double-stranded RNA, and share a 350-amino acid 2–5A synthetase-like domain that is thought to be essential for catalytic activity (54, 56). OAS is an integral mediator of the antiproliferative effects of IFNs, as antisense ablation of OAS in NIH/3T3 cells reversed IFN-mediated growth inhibition and resulted in anchorage-independent growth of colonies in soft agar (57). Additionally, ectopic expression of the 42-kDa OAS in HL-60 myeloid leukemic cells resulted in cell growth arrest and the appearance of a myeloid differentiation marker (58). Thus, OAS may also play a more general role in normal cell growth and differentiation in addition to its well characterized role in mediating an antiviral response.

The physical interaction of OAS with the PRL-R implies a potential role of this enzyme in PRL-R signal transduction. Our studies suggest that OAS inhibits PRL-R signaling to Stat1, resulting in reduced Stat1 binding to the IRF-1 GAS element (Fig. 4Go, A–D). Importantly, this decrease in Stat1 DNA binding is not due to degradation of Stat1 protein (Fig. 4EGo). This reduction in PRL-inducible Stat1 binding to the IRF-1 GAS element corresponds with a concomitant decrease in PRL activation of the IRF-1 promoter (Figs. 2Go and 3Go). How OAS mediates the reduction in Stat1 DNA binding activity and whether there is a direct association of OAS with Stat1 are as yet unclear. Stat1 DNA binding and transcriptional activities are modulated by its interaction, either directly or indirectly, with other nuclear factors including Stat2 (59), p48 (60), USF-1 (61), Sp1 (62), and nuclear factor-{kappa}B (63), as well as the coactivator CBP/p300 (64, 65, 66). Stat1 also interacts with cytoplasmic factors that can modulate its transcriptional activities. One such factor is Stat-interacting protein (STIP) (67), which is thought to facilitate phosphorylation of Stat factors by JAK tyrosine kinases. Another factor is the N-myc interacting protein (Nmi) (68) that exists in the cytoplasm but translocates into the nucleus upon cytokine stimulation and enhances Stat factor function. Interestingly, a third protein recently found to associate with Stat1 is protein kinase R (PKR), an enzyme that inhibits protein synthesis in the IFN antiviral pathway (69). PKR physically associates with Stat1, and their dissociation increases Stat1 binding in response to IFN stimulation. These observations indicate that PKR and OAS, two enzymes critical for IFN-mediated inhibition of cellular metabolism, may also serve additional roles in cytokine receptor signal transduction.

In contrast to its effects on PRL-mediated Stat1 binding to the IRF-1 GAS element, OAS did not alter Stat5 DNA binding activity (Fig. 4BGo) or affect PRL signaling to the ß-casein promoter (Fig. 2Go, B and D). Different regions of the ICD of the PRL-R are involved in the recruitment of Stats in response to PRL stimulation. Tyrosine 580 (equivalent to Y382 in the Nb2 form of the PRL-R) is important for recruitment of Stat5 to the PRL-R (70), while both tyrosines 309 and 580 (equivalent to Y309 and Y382 in the Nb2 PRL-R) are required for the recruitment of Stat1 to the PRL-R (14). A working model is presented to depict potential interactions of OAS with the ICD of the PRL-R (Fig. 5Go). It is interesting to speculate that OAS interacts with the region of the PRL-R containing Y309 and differentially alters Stat activation along the PRL-R signaling pathway. OAS affects Stat1 recruitment at Y309, but not Stat1 or Stat5 interaction at Y580 in the long form of the PRL-R or Y382 in the Nb2 form of the PRL-R. In this way, PRL-inducible Stat1-regulated target genes such as IRF-1 would be partially affected by OAS interaction with the PRL-R, while the expression of PRL-inducible Stat5-regulated target genes such as ß-casein would not be altered. This model is supported by the observation that Stat1 DNA binding (Fig. 4Go) and transcriptional activities (Figs. 2Go and 3Go) are reproducibly reduced by about 40%, indicating only partial inhibition by OAS after PRL stimulation, while Stat5 DNA binding (Fig. 4BGo) and function (Fig. 2Go, B and D) remain unaltered. However, this model does not preclude the possibility that at higher OAS concentrations, additional sites of OAS interactions with the PRL-R may be found. This notion is supported by the observation that 2 µg OAS led to a near-complete inhibition of PRL-inducible Stat1 DNA binding at the IRF-1 GAS (Fig. 4DGo), in the absence of any effect on Stat1 protein levels (Fig. 4EGo). Our interpretation is that overexpression of the 42-kDa OAS either reduced PRL-inducible Stat1 interactions with both Y309 and Y580 in the long PRL-R or resulted in inhibition of cellular metabolism that is independent of PRL-R signaling. Whether Stat5 activation via Y580 in the long PRL-R is affected by higher OAS concentrations should help distinguish between these possibilities. Since both the long and Nb2 forms (data not shown) of the PRL-R interact with OAS by the yeast two-hybrid genetic assay and by coimmunoprecipitation in unstimulated COS-1 cells (Fig. 1CGo), it is likely that the PRL-R/OAS interaction does not require receptor activation. Additionally, since both PRL-R forms mediate OAS inhibition of PRL signaling to the IRF-1 promoter (Fig. 2Go, A and B, and Fig. 3Go for the long PRL-R; and Fig. 2Go, C and D, for the Nb2 PRL-R), it appears that the 198-amino acid in-frame deletion of the long PRL-R, which generates the Nb2 PRL-R, is not involved in OAS interaction. Mapping of the precise PRL-R domain that is interacting with OAS should verify this hypothesis.



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Figure 5. Working Model of OAS Interaction with the PRL-R

OAS was identified as a PRL-R interacting protein in a yeast two-hybrid screen using the ICD of the long form of the PRL-R as bait. OAS appears to interact constitutively with a region around tyrosine 309 (Y309) in the ICD of both the long and Nb2 forms of the PRL-R. This interaction affects PRL-inducible Stat1 recruitment to Y309, reduces Stat1 DNA binding activity, and reduces the expression of Stat1-regulated target genes such as IRF-1. OAS inhibition is not complete as PRL-inducible Stat1 can be recruited via Y580 in the long PRL-R (or its equivalent Y382 in the Nb2 PRL-R). On the other hand, OAS does not affect PRL-inducible Stat5 recruitment to Y580. Thus, PRL signaling to Stat5-regulated target gene such as ß-casein is not affected by OAS interaction with the PRL-R. Only two of the tyrosine residues in the ICD of the PRL-R important for Stat1 and Stat5 signaling are shown. 198 aa, Intracellular region of the long form of the PRL-R that is deleted in the Nb2 PRL-R (9 ); 1, Stat1; 5, Stat5.

 
Our interpretation is that OAS through its association with the PRL-R regulates some aspects of PRL-R-mediated signaling to the immediate early gene IRF-1. It is interesting to speculate that OAS may contribute to a mechanism to down-regulate Stat1-mediated PRL induction of IRF-1 gene transcription. Several other molecules have recently been identified to modulate cytokine receptor signaling at multiple levels along the signaling pathway. Both SHP-1 tyrosine phosphatase (71, 72) and SOCS (for suppressor of cytokine signaling, which is also known as CIS/JAB) can inhibit JAK2 tyrosine kinase activity (73, 74). SOCS-1 (suppressor of cytokine signaling) associates with JAK2 and inhibits its activation by PRL stimulation (75). Interestingly, SOCS-2 appears to interact directly with the PRL-R, block SOCS-1 activity, and restore JAK2 activation and PRL signaling (75). A nuclear phosphatase is thought to dephosphorylate and thereby inactivate Stat factors in the nucleus (76). PIAS (protein inhibitor of activated Stat) interacts with Stat proteins and inhibits their function in response to cytokine signaling (77, 78). Recently, nitric oxide (NO) was found to modulate Tyk2 and Stat4 activity in NK cells (79). Thus, distinct molecules can impact cytokine receptor signaling through interaction with or modulation of the activity of Stat factors. Whether OAS is affecting PRL-R recruitment of Stat1, Stat1 activation by JAK2 tyrosine kinase, Stat1 interactions with other cytoplasmic and/or nuclear factors, or nuclear translocation of Stat1, all of which can result in reduced Stat1 binding to the IRF-1 GAS element, is under investigation.

An OAS-related protein, p56 (80, 81), which shares a highly conserved N terminus but a distinct C terminus with other OAS proteins, has recently been described. Interestingly, the p56 OAS-related protein does not have the ability to form 2–5A oligomers and is localized in the nucleolus (81), suggesting that it may have functions distinct from activation of the RNase L pathway and RNA metabolism. In combination with the data reported here, these studies imply that OAS and OAS-related proteins may play a more significant role in cellular signaling and metabolism than previously appreciated. It will be interesting to determine whether OAS or the OAS-related proteins associate with other cytokine receptors and thereby function as a general mediator of cytokine signaling.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DNA Constructs
For the yeast two-hybrid screens, the entire ICD of the long form of the PRL-R was generated by PCR from the rat PRL-R cDNA (82) using primers corresponding to the first five and last five amino acids, respectively, of the ICD: 5' sense (EcoRI underlined)-ggaattcAAGGGCTATAGCATG and 3' antisense (PstI underlined)-aactgcagGTGAAAGGAGTGCAT(83). The ICD PCR fragment was confirmed by sequencing, digested with EcoRI and PstI, and cloned into the pGBT9 yeast vector to use as bait to interact with pACT yeast clones derived from a human B cell cDNA library (CLONTECH Laboratories, Inc. Palo Alto, CA) (42). For the in vitro interaction assay, the EcoRI-PstI PRL-R ICD was subcloned into the MBP bacterial expression vector pMALp (pMALp-ICD) (New England Biolabs, Inc., Beverly, MA). The human 42-kDa OAS cDNA in pACT was released with XhoI, subcloned into pBK-CMV (Promega Corp., Madison, WI), released by NotI and BamHI digests, and subcloned into pcDNA3.0 (pcDNA3-OAS) (Invitrogen, San Diego, CA) for in vitro translation and reporter transfection studies.

Yeast Two-Hybrid Screen
The PRL-R ICD in the yeast pGBT9 vector was cotransformed with the human B cell cDNA library in pACT vector into the HF7C yeast strain as suggested by the manufacturer (CLONTECH Laboratories, Inc.) (42). Protein-protein interaction was scored by the ability of the transformed yeast colonies to grow on stringent selection medium that was deficient in leucine, tryptophan, and histidine but was supplemented with 50 mM 3-aminotriazole (42). Colonies that survived this growth selection were screened for ß-galactosidase activity.

MBP Fusion Protein Pull-Down Assay
Approximately 200 µg MBP-PRL-R ICD or control MBP were incubated with amylose resins (New England Biolabs, Inc.) at 4 C for 30 min. The resins were then sequentially washed with buffer A (10 mM sodium phosphate, pH 7.2, 0.5 M NaCl, 1 mM sodium azide), twice with buffer B (20 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP40, and 0.5% milk), and twice with buffer C (20 mM HEPES, pH 7.9, 60 mM NaCl, 1 mM dithiothrietol, 6 mM MgCl2, 0.1 mM EDTA, and 10% glycerol). Between 10 and 25 µl in vitro-translated [35S]methionine-labeled OAS (see below) along with 200 µl buffer C were incubated with the resins for 1 h at 4 C. The resins were washed twice each with buffer B and buffer C at 4 C. Bound proteins were resolved by 10% SDS-PAGE and analyzed by autoradiography.

TNT Translation of OAS
The 42-kDa OAS was transcribed and translated in vitro using the T3 TNT-coupled reticulocyte lysate system (Promega Corp.). T7 RNA polymerase (Promega Corp.), 1 µg pcDNA3-OAS, and 40 µCi [35S] methionine (1150 Ci/mmol, ICN Biochemicals, Inc., Irvine, CA) were used in 50 µl of TNT reaction as previously described (84).

Coimmunoprecipitation
COS-1 cells (1.6 x 106/10 cm plate) were transfected with lipofectamine as described below with 1 µg of Flag-tagged mouse 42-kDa OAS (48) (gift of Ganes Sen, Cleveland Clinic Foundation, Cleveland, OH) and either 1 µg of the long form of the PRL-R or 4 µg of the Nb2 form of the PRL-R. After 24 h, cells were lysed in PBS containing 0.5% Triton X-100, and insoluble proteins were removed by centrifugation. Anti-Flag-antibodies coupled to agarose beads (Eastman Kodak Co., Rochester, NY) were added to the supernatant and rocked at room temperature for 2.5 h. The beads were washed three times in PBS containing 0.1% Triton X-100 and boiled in SDS sample buffer, and the bound proteins were resolved by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The bound proteins were immunoblotted with polyclonal anti-PRL-R ICD peptide antibodies (R123) (14) (1:400 dilution), followed by donkey antirabbit antibodies conjugated to horseradish peroxidase (1:3000 dilution) (Bio-Rad Laboratories, Inc. Hercules, CA), which was detected by enhanced chemiluminescence (ECL) as suggested by the manufacturer (Amersham Pharmacia Biotech, Piscataway, NJ). A parallel blot was probed with anti-B peptide antibodies directed against human 42-kDa OAS (47) (1:400 dilution) to verify equal Flag-tagged OAS expression (data not shown).

Transient Transfection and CAT Assays
COS-1 cells (3 x 105 cells per well) were plated in a six-well tissue culture dish overnight in DMEM containing 10% FBS (Life Technologies, Gaithersburg, MD). Transient transfections were performed using LipofectAMINE (Life Technologies). Briefly, cells were rinsed with DMEM, and then incubated with the LipofectAMINE-DNA mixture in a total volume of 1 ml DMEM. DNA concentrations were as follows: 1 µg long or Nb2 form of the PRL-R (82), 1 µg Stat1 (14) or Stat5b (21), 1 µg pcDNA3-OAS or control pcDNA3.1 vector, and various promoter reporter constructs. These include 0.2 µg 1.7-kb IRF-1-CAT (49), 0.2 µg 0.2-kb IRF-1-CAT (49), 0.5 µg 3C GAS-TK-CAT (14), or 1 µg 2.3-kb ß-casein-CAT (21, 50). After 5 h, the LipofectAMINE-DNA mixture was removed and replaced with DMEM containing 1% donor horse serum (JRH Biosciences, Lenexa, KS) for 18–24 h. Cells were stimulated with 100 ng/ml ovine PRL (NIDDK oPRL-20) for 24 h, washed with PBS, and harvested using reporter lysis buffer (Promega Corp.). The 1.0 µg exogenous OAS did not appreciably alter cellular growth rates during the 48-h duration of the transfection experiments (data not shown). Thus, 1.0 µg OAS was routinely used for promoter transfection experiments while 2.0 µg OAS was only used for shorter duration EMSA experiments (see below). Equal volumes of protein extracts were assayed for CAT enzyme activity by incubating with 5 µl 5 mg/ml n-butyryl-coenzyme A (Promega Corp.) and 3 µl [14C]chloramphenicol (50 mCi/mmol, NEN Life Science Products, Boston, MA) for 3–4 h at 37 C. After xylene extractions, CAT activity was determined by liquid scintillation counting. Data were expressed either as counts per min [14C]chloramphenicol converted/µg protein, or as a percent of the maximum counts per min/µg protein values, which were obtained from the empty vector + PRL samples, to compare CAT activity between experiments. Data were analyzed and plotted using the Microcal Origin 4.1 graphics program (Microcal Software, Northampton, MA).

EMSA
COS-1 cells (1.7 x 106/10 cm plate) were transfected as described above, with 1 µg each of the long PRL-R, Stat1, and pcDNA3-OAS or control pcDNA3.1 vector unless otherwise indicated. Cells were stimulated with 100 ng/ml oPRL for the indicated times, and whole cell lysates were prepared. Briefly, the cells were resuspended in 300 µl ice-cold buffer A [10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mMM KCl, 2 mM dithiothreitol (DTT)] for 5 min, followed by vortexing in 30 µl ice- cold buffer C (20 mM HEPES, pH 7.9, 20% glycerol, 0.55 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 2 mM DTT) for 20 min. After centrifugation at 14,000 rpm for 10 min, the supernatant was employed for EMSA. A 27-mer rat IRF-1 GAS oligo, top strand 5'-AACAGCCTGATTTCCCCGAAATG-3', and bottom strand, 5'-TCATCATTTCGGGGAAATCAGGCTGTT-3' (Bio-Synthesis, Inc., Lewisville, TX) was labeled with [{alpha}-32P]dATP and [{alpha}-32P]dTTP as described previously (21). Two micrograms of COS extract were incubated with 0.3–1.0 ng GAS probe (3–5 x 105 cpm) in a reaction mixture (12 mM HEPES pH 7.9, 0.1 mM EDTA, 5 mM MgCl2, 10% glycerol, 60 mM KCl, 1 mM DTT, 2 µg of poly (dI-dC), and 40 ng pdN6) for 20 min at room temperature. For competition and antibody supershift experiments, the extracts were preincubated with 50x molar excess of unlabeled GAS probe, monoclonal anti-Stat1 antibodies (G16920, Transduction Laboratories, Inc., Lexington, KY), monoclonal anti-Stat6 antibodies (anti-IL-4Stat, S25420, Transduction Laboratories, Inc.), or affinity-purified polyclonal anti-Stat5b peptide-specific antibodies (21) for 20 min on ice before the addition of labeled GAS probe. Samples were resolved on a 5% nondenaturing acrylamide gel by electrophoresis for 2 h at 16 mA using 0.25x Tris-Borate-EDTA buffer. Gels were dried, analyzed by autoradiography, and scanned using Storm960 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

Western Blot Analysis
COS-1 cells (3 x 105/well) were transfected as described above with 1 µg of the long PRL-R and Stat1, and the indicated amounts of pcDNA3-OAS (0–2 µg), with pcDNA3.1 vector used to equalize the amount of DNA transfected for each condition. After stimulation with 100 ng/ml oPRL for the indicated times, the cells were harvested by gently scraping in 1x PBS and pelleted. Cell pellets were lysed in 1 ml of RIPA buffer (50 mM Tris, pH 7.4, 0.5% NP-40, 0.2% sodium deoxycholate, 100 mM NaCl, 1 mM EGTA, 1 mM AEBSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM DTT, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 1 mM NaF) for 30 min at 4 C. Ten microliters of the cell lysates were run on an 8% SDS-PAGE and transferred to Immobilon-P membrane (Millipore Corp., Bedford, MA). For Western blotting, polyclonal anti-Stat1 antibodies (G16930, Transduction Laboratories, Inc.) were used (1:1000 dilution) followed by horseradish peroxidase-conjugated donkey antirabbit antibodies (1:2000 dilution) (Amersham Pharmacia Biotech), which were detected using ECL (Amersham Pharmacia Biotech).


    ACKNOWLEDGMENTS
 
We would like to thank Dr. Gyorgyi Horvath for yeast two-hybrid cloning of OAS, Drs. Sue-Hwa Lin, Sophia Tsai, and Jeff Rosen for helpful discussions and comments, Rhonda Chaplin and Kerry Sieger for excellent technical assistance, Dr. Sue-Hwa Lin for assistance with Co-IPs, and Dr. Ganes Sen for providing Flag-tagged OAS.


    FOOTNOTES
 
Address requests for reprints to: Dr. Li-yuan Yu-Lee, Department of Medicine, Baylor College of Medicine, Houston, Texas 77030.

This work was supported in part by an NIH Training Grant T32-AI07495 (K.M.M.), Heilig-Meyers Foundation Fellowship (M.L.B.), American Cancer Society Grant BE-425 (L.-y. Y.-L.), and NIH RO1 DK-53176 (L.-y. Y.-L.).

1 Current address: Department of Immunology, M. D. Anderson Cancer Center, Houston, Texas 77030. Back

2 Co-first authors. Back

Received for publication July 13, 1999. Revision received November 5, 1999. Accepted for publication November 10, 1999.


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 ABSTRACT
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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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