JAK2 and STAT5, but not JAK1 and STAT1, Are Required for Prolactin-Induced ß-Lactoglobulin Transcription

Yulong Han, Diane Watling, Neil C. Rogers and George R. Stark

Department of Molecular Biology (Y.H., G.R.S.) Research Institute The Cleveland Clinic Foundation Cleveland, Ohio 44195
Imperial Cancer Research Fund (D.W., N.C.R.) Lincoln’s Inn Fields London WC2A 3PX, UK


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Several different Janus kinases (JAKs) and signal transducers and activation of transcription (STATs) have been implicated in mediating the biological responses induced by PRL, based on their ligand-dependent tyrosine phosphorylation and activation. However, these criteria alone do not prove that a particular JAK or STAT is essential for signal transduction. We have used mutant cell lines defective in JAK1, JAK2, or STAT1 to examine their roles in PRL-dependent signaling. JAK2 is absolutely required for PRL-dependent phosphorylation of the receptor, activation of STATs, and induction of ß-lactoglobulin. Wild type, but not kinase-negative JAK2, restores all responses to PRL in JAK2-defective cells, suggesting that JAK2 function, not merely the protein, is required. In contrast, JAK1, which is phosphorylated in response to PRL, is not required for any of these functions. Although STAT1 homodimers do form in response to PRL, no defect in PRL-dependent signaling is apparent when STAT1 is missing, suggesting that STAT5, which is strongly activated in response to PRL, is primarily responsible for driving the expression of PRL-responsive genes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Janus kinases (JAKs) and signal transducers and activators of transcription (STATs), originally identified as components of interferon-dependent signaling pathways (1), have also been shown to mediate many of the biological responses induced by cytokines and growth factors (2, 3). To date, four mammalian JAKs (JAK1, JAK2, JAK3, and TYK2) (4, 5, 6, 7, 8, 9) and seven STATs (STATs 1 to 4, 5a, 5b, and 6) have been cloned (10, 11, 12, 13, 14, 15, 16). JAK and STAT family members have also been identified in Drosophila (17, 18, 19, 20, 21, 22). Ligand binding leads to tyrosine phosphorylation and activation of JAKs, which are usually bound to the receptors. Phosphorylation of receptor subunits and STATs then ensues. JAKs may also activate other signaling components (23), and STATs may be activated by the intrinsic kinases of some growth factor receptors (24) or by nonreceptor kinases (25, 26, 27, 28).

PRL, a polypeptide hormone from the anterior pituitary gland, regulates several different physiological responses, especially milk gene expression, and also has a role in modulating immune responses (29, 30). PRL binds to a cell-surface receptor, PRLR, which belongs to a class of cytokine and growth factor receptors lacking intrinsic kinase activity (31, 32). Binding of PRL stimulates rapid, transient tyrosine phosphorylation of several intracellular proteins, including the receptor and a subset of JAKs and STATs (14, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45), and also leads to the transcriptional activation of PRL-responsive milk protein genes (46, 47) and the interferon-regulatory factor 1 (IRF-1) gene (48, 49, 50). JAK2 (35, 36, 37, 38, 39, 40, 41) and JAK1 (35) are both bound to PRLR and become phosphorylated upon ligand binding. STATs 1, 3, and 5 are activated in response to PRL and form homo- and heterodimers, which bind to {gamma}-interferon-activated sequence (GAS) elements in the promoters of milk protein and IRF-1 genes (41, 42, 43, 44, 45).

Recent studies of other pathways reveal that the JAKs and STATs activated in response to ligand binding may not always be essential for signaling. For example, interleukin-6 (IL-6) induces the phosphorylation of several different JAKs, with a pattern that varies in different cells. However, only JAK1 is vital for IL-6-dependent phosphorylation of the gp130 receptor subunit, STAT activation, and transcriptional induction of the IRF-1 gene (51). GH also activates both JAK1 and JAK2, but only JAK2 is required for phosphorylation and activation of the receptor, the signal transducer Shc, and the STATs (23). Platelet-derived growth factor induces phosphorylation of JAK1, JAK2, and TYK2, but deletion of each of these kinases singly does not affect STAT activation (52). Epidermal growth factor (EGF) stimulates phosphorylation of JAK1 and STATs 1 and 3, but JAK1 is not required for STAT activation, and neither JAK1 nor STAT1 is required for induction of the c-fos gene by EGF (24). Furthermore, STAT1-deficient mice lack all responses to interferons (IFNs), as expected from the prior work with STAT1-deficient human cells (53), but the mice respond normally to GH, EGF, and IL-10, which activate STAT1 in cell lines, arguing against any essential role for STAT1 in these pathways (54).

Here, by using PRLR-transfected cell lines lacking JAK1, JAK2, or STAT1, we show that JAK2 is essential for PRL-dependent STAT activation, receptor phosphorylation, and ß-lactoglobulin induction and that JAK1 and STAT1 are not.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of Functional PRLR
The parental cell lines 2C4 and 2fTGH (not shown) and the mutant cell lines derived from them do not express endogenous PRLR, as determined by use of the FACScan (Fig. 1Go, top left) and Western analyses (data not shown). Therefore, each cell line was transfected with a cDNA encoding the long form of rabbit PRLR. After selection for stable transfectants, cells from individual clones were scanned to identify those that expressed high levels of PRLR. FACScan (Fig. 1Go) and Western analyses (data not shown) revealed that the cells used in all experiments expressed similar levels of PRLR. PRL stimulation of 2C4 cells transfected with rabbit PRLR (2C4/PRLR) induced the phosphorylation on tyrosine of several cellular proteins with apparent molecular masses of 90–130 kDa (data not shown), indicating that the transfected cells did express functional receptors.



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Figure 1. Expression of PRLR in Parental and Mutant Cell Lines

FACScan analysis (10,000 data points) of cell surface PRLR expression in the cell lines 2C4/PRLR, U4C/PRLR, {gamma}2A/PRLR, and U3A/PRLR (thick solid lines). Profiles of unstained 2C4/PRLR (dotted line) and stained 2C4 (thin solid line) cells are included for comparison.

 
PRL-Dependent Activation of STATs
Previously, PRL has been shown to stimulate phosphorylation on tyrosine of STATs 1, 3, and 5. To examine STAT phosphorylation in response to PRL treatment of the transfected cells listed above, immunoprecipitations with antibodies to STATs 1 and 5 were performed, using extracts of cells treated with PRL or left untreated. Cells treated with IFN{gamma} in parallel were used as controls. PRL induces strong phosphorylation of STAT5 on tyrosine in 2C4/PRLR cells (Fig. 2AGo, lane 2), but STAT1 phosphorylation was not detected using the same cell extracts (Fig. 2BGo, lane 2). As expected, IFN{gamma} stimulates tyrosine phosphorylation of STAT1 (Fig. 2BGo, lane 3), but not STAT5 (Fig. 2AGo, lane 3). In U3A/PRLR cells, the intensity of PRL-dependent STAT5 phosphorylation (Fig. 2AGo, lane 11) was similar to the intensity in control 2C4/PRLR cells (lane 2), suggesting that PRL-dependent STAT5 phosphorylation is independent of STAT1. Similar results were observed by electrophoretic mobility shift assay (EMSA) (Fig. 3Go, lanes 2 and 12). Activation of STAT3 by PRL has been observed in some cell lines (see Discussion) but was not detected in our cells by either immunoprecipitation (data not shown) or EMSA (see below).



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Figure 2. Stimulation of STAT Phosphorylation by PRL or IFN{gamma}

The cell lines 2C4/PRLR, U4C/PRLR, {gamma}2A/PRLR, U3A/PRLR, {gamma}2A/PRLR/JAK2, and {gamma}2A/PRLR/JAK2KE ({gamma}2A/PRLR complemented with murine JAK2 or JAK2 KE mutant cDNA) were untreated (-) or treated with 1 µg/ml PRL or 500 IU/ml IFN{gamma} for 15 min at 37 C. The tyrosine phosphorylation of STATs was analyzed by immunoprecipitating them from cell lysates, using anti-STAT5 (A) or anti-STAT1 (B), followed by Western blotting and analysis with antiphosphotyrosine antibodies (A). The transfers were stripped and reprobed with anti-STAT sera (B).

 


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Figure 3. EMSA Analysis of PRL-Induced Binding to a ß-Casein GAS Probe

Extracts were prepared from the cell lines 2C4/PRLR, U4C/PRLR, {gamma}2A/PRLR, {gamma}2A/PRLR/JAK2, {gamma}2A/PRLR/JAK2KE, or U3A/PRLR, untreated (lane 1), treated with PRL (lanes 2, 4, 6, 8, 10, 12), or treated with IFN{gamma} (lanes 3, 5, 7, 9, 11, 13) for 15 min at 37 C. The positions of the PRL-inducible complexes A and B are indicated.

 
EMSA was used to characterize the DNA-binding properties of the phosphorylated STAT proteins induced by PRL. At least two PRL-inducible complexes (A and B) were detected in extracts of parental 2C4/PRLR cells with a GAS probe from the promoter of the PRL-responsive ß-casein gene (Fig. 3Go, lane 2). The intensity of complex A was at least 10 times higher than that of complex B. Complex B migrated similarly to the IFN{gamma}-induced complex (Fig. 3Go, lane 3) and was not observed in U3A/PRLR cells treated with PRL or IFN{gamma} (Fig. 3Go, lanes 12 and 13). The formation of complex B was completely competed by various unlabeled GAS elements, but complex A was completely competed only by the ß-casein GAS. Unrelated DNA elements did not affect either complex (data not shown). To analyze the STATs present in these complexes, antibody supershift experiments were performed. Complex B was supershifted only by anti-STAT1 (Fig. 4Go, lanes 2 and 9), indicating that this complex probably consists of STAT1 homodimers bound to the probe. STAT1–5 heterodimers were not observed: Complex A, observed only in cells treated with PRL (Fig. 3Go, lane 2) and not IFN{gamma} (Fig. 3Go, lane 3), was supershifted only by anti-STAT5 (Fig. 4Go, lane 5). Antisera to STATs 2, 3, and 6 and preimmune serum had no effect (Fig. 4Go, lanes 3, 4, 6, and 7).



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Figure 4. Antibody Supershift Analysis of PRL-Induced STAT Binding to a ß-Casein GAS Probe

Whole-cell extracts from PRL-treated 2C4/PRLR (lanes 1–7) or {gamma}2A/PRLR/JAK2 cells (lanes 8–10) were analyzed. The STATs present in complexes were evaluated with antisera to STAT1 (lanes 2 and 9), STAT2 (lane 3), STAT3 (lane 4), STAT5 (lanes 5 and 10), STAT6 (lane 6) or with preimmune serum (PI, lane 7). The positions of the PRL-induced complexes A and B, observed in both cells, are indicated.

 
JAK2 Is Essential for the PRL-Dependent Activation of STATs
The PRLR lacks an intrinsic kinase domain and signals through receptor-associated kinase(s). JAK2 is strongly phosphorylated in response to PRL in our cells, and JAK1 is phosphorylated very weakly, about 10% as strongly as in response to IFN{gamma} (data not shown). JAK1 has been shown by some (35) but not others (36, 37, 55) to be activated in response to PRL in other cell types, suggesting that JAKs 1 and 2 might both be involved in PRL-induced signaling. STAT phosphorylation and activation in response to PRL were normal in U4C/PRLR cells (lacking JAK1, Fig. 2AGo, lane 5, and Fig. 3Go, lane 4) but were not observed in {gamma}2A/PRLR cells (lacking JAK2, Fig. 2Go, lane 8, and Fig. 3Go lane 6). Wild type murine JAK2 (Fig. 2AGo, lane 14, and Fig. 3Go, lane 8), but not kinase-negative JAK2 (Fig. 2AGo, lane 17, and Fig. 3Go, lane 10), restored PRL-induced STAT phosphorylation and DNA-binding activity in {gamma}2A/PRLR cells. These results show that functional JAK2, but not JAK1, is absolutely required for PRL-dependent STAT activation. As expected, both JAK1 and JAK2 are required for STAT1 activation by IFN{gamma} (Fig. 2BGo, lanes 6 and 9; Fig. 3Go, lanes 5 and 7).

PRL-Induced Tyrosine Phosphorylation of the PRLR Requires JAK2
Tyrosine phosphorylation of the PRLR is required for PRL-dependent cell proliferation and gene activation (33, 34, 35, 36, 37, 38). Although both JAK1 and JAK2 become phosphorylated, it is not clear whether both are required for receptor phosphorylation. To examine this issue, immunoprecipitates prepared from cell lysates of PRLR-transfected cells with anti-PRLR antibody S46 were analyzed with antiphosphotyrosine antibodies. In response to PRL, the receptor is phosphorylated on tyrosine in 2C4/PRLR and U4C/PRLR cells (Fig. 5Go, lanes 4 and 6), but not in {gamma}2A/PRLR cells (lane 8). (The difference in PRLR expression between lanes 4 and 6 of Fig. 5AGo and B was not seen in independent repeats of this experiment.) Wild type murine JAK2 (lane 10), but not kinase-negative JAK2 (lane 12), restored receptor phosphorylation in {gamma}2A/PRLR cells, to a level similar to that of parental 2C4/PRLR cells. As expected, no signal was observed with 2C4 cells, which lack the PRLR (lane 1) and no phosphorylated PRLR was detected in 2C4/PRLR cells treated with IFN{gamma}. These data show that JAK2, but not JAK1, is required for PRL-dependent receptor phosphorylation.



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Figure 5. Requirement for JAK2 in PRL-Dependent PRLR Phosphorylation

2C4, 2C4/PRLR, U4C/PRLR, {gamma}2A/PRLR, {gamma}2A/PRLR/JAK2, or {gamma}2A/PRLR/JAK2KE cells were untreated (lanes 3, 5, 7, 9, 11) or treated with PRL (lanes 1, 4, 6, 8, 10, 12) or IFN{gamma} (lane 2) for 15 min at 37 C. Cell lysates were analyzed by immunoprecipitation with a polyclonal antibody to rabbit PRLR, followed by Western blotting and analysis with antiphosphotyrosine antibodies (A). The blots were stripped and reprobed with anti-PRLR sera (B).

 
JAK2-Dependent Stimulation of ß-Lactoglobulin Expression
Stimulation of responsive cells by PRL activates transcription of several genes, including ß-lactoglobulin. Since JAK2, but not JAK1, is required for PRL-dependent activation of STAT proteins and phosphorylation of the PRLR, it was important to evaluate the role of JAKs in PRL-induced transcriptional activation. ß-Lactoglobulin promoter/chloramphenicol acetyltransferase (CAT) constructs were stably transfected into 2C4/PRLR, U4C/PRLR, or {gamma}2A/PRLR cells. In the absence of JAK2, CAT induction was almost absent in response to PRL (Fig. 6Go, lane 11), but in the absence of JAK1 (lane 14), CAT induction was similar to that in parental 2C4/PRLR cells (Fig. 6Go, lane 5). Wild type murine JAK2 substantially restored CAT induction by PRL in {gamma}2A/PRLR cells in a transient transfection assay (lane 8). Surprisingly, although IFN{gamma} induced strong STAT1 activation (Figs. 2Go and 3Go), it did not induce CAT activity in 2C4/PRLR cells (Fig. 6Go, lane 3), showing that the STAT1 homodimer induced by IFN{gamma} cannot drive gene expression from the ß-lactoglobulin GAS element.



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Figure 6. PRL-Dependent Activation of ß-Lactoglobulin Expression in Mutant Cell Lines

Cells were transfected stably (2C4/PRLR, U4C/PRLR, {gamma}2A/PRLR, U3A/PRLR) or transiently ({gamma}2A/PRLR/JAK2) with the ß-lactoglobulin/CAT construct pBJ23. CAT assays were performed with extracts from serum-starved cells, untreated (lanes 1, 4, 7, 10, 13) or treated with PRL (lanes 2, 5, 8, 11, 14) or IFN{gamma} (lanes 3, 6, 9, 12, 15). Values are expressed as percent chloramphenicol conversion and as fold induction calculated from the basal level activities (representative of three experiments).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previous observations have shown that PRL activates the rapid and transient tyrosine phosphorylation of JAK family members, either JAK2 in Nb2 cells (36, 37, 38, 39, 40, 41) or JAK2 and JAK1 (weakly) in BAF-3 cells (35). Nb2 cells are a rat T cell lymphoma line expressing a truncated form of the PRLR with a deletion of 198 amino acids in the middle of the cytoplasmic domain (56); BAF-3 and 2C4 are an IL-3-dependent pro-B murine lymphoid line and a human fibrosarcoma line, respectively, and both express the full-length PRLR (35). It is not yet clear whether activation of a different set of JAKs by PRL in these cells is due to cell type differences or to the different types of PRLRs that are present. When we transfected the long form of PRLR into human fibrosarcoma 2C4 cells, the pattern of JAK phosphorylation in response to PRL was similar to that in BAF-3 cells (data not shown). The PRL-induced phosphorylation of JAK1 has been detected only in cells expressing the full-length receptor (35), and the activation of JAK1 by GH requires regions of that receptor’s cytoplasmic domain beyond the membrane-proximal region that usually associates with JAKs (Y. Han and G. R. Stark, unpublished data). These results suggest that the activation by PRL or GH of JAK1 is different from that of JAK2. In addition, the pattern of activation of several different JAKs by some cytokines is distinct in different cell lines (57).

We and others have demonstrated recently that a particular JAK may not be essential for STAT-mediated signal transduction even if it becomes phosphorylated and activated upon ligand binding (23, 24, 51, 52). Therefore, we examined the functions of JAK1 and JAK2 in PRL signaling in mutant cell lines lacking these kinases. Complete defects in the PRL-dependent activation of STATs, phosphorylation of the receptor, and induction of ß-lactoglobulin were observed in {gamma}2A/PRLR cells lacking JAK2, but not in U4C/PRLR cells lacking JAK1, revealing that JAK2 but not JAK1 has an obligatory role in PRL-dependent signaling. Wild type JAK2, but not a kinase-negative mutant, restores the response, indicating that active JAK2, and not merely the protein, is required. However, a requirement for JAK1 in other PRL-dependent signaling cannot be excluded insasmuch as JAKs can function in alternative pathways (23).

STAT5 was originally identified as a PRL-activated transcription factor involved in the expression of milk proteins in sheep (14). Subsequently, it was shown that mice have two STAT5 homologs, 5a and 5b (58, 59, 60). Two forms of human STAT5 have also been identified (61). Both STATs 5a and 5b bind to a GAS element in the ß-casein gene promoter and function in signaling in response to IL-3, IL-12, and PRL (59, 60, 61). STATs 5a and 5b heterodimers were recently observed (62), but STAT5 heterodimers with other STATs have not been reported. Therefore, the activated STAT5 we have observed could represent either a STAT5b homodimer, a STAT5a-5b heterodimer, or both, inasmuch as the antibody used is specific for STAT5b and does not cross-react with other STATs.

In PRL-treated rat Nb2 T-lymphocytes, activation of STAT1 has been observed by immunoprecipitation (40). STAT1 was activated much less than STAT5 in our PRLR-transfected cells, and phosphorylated STAT1 was detected only in the sensitive EMSA. As a control, STAT1 was strongly activated by IFN{gamma} in the same cells. These observations reveal that IFN{gamma} prefers to activate STAT1, and PRL prefers STAT5 (1, 2, 3, 14). Less activation of STAT1 by PRL in 2C4/PRLR cells compared with Nb2 cells seems not to be due to a difference in the amount of the STAT1 protein because the level is high in both cases, with strong activation by IFN{gamma} (Figs. 2Go and 3Go). The difference could be due to the fact that Nb2 cells have an abnormally high response to PRL, which may be related to their truncated form of the receptor (35, 56). Activation of STAT3 by PRL has been observed in cos-1 cells cotransfected with STAT3 and PRLR cDNAs (42) and in 32D premyeloid cells (63), but not in our cells, even when EMSA was performed with several different GAS elements (data not shown). Different patterns of STAT activation in different cell types have been observed with several cytokines and also with growth factors (23, 24, 64), raising the possibility that selective activation of specific STAT subsets can contribute to the regulation of specific subsets of genes in a cell type-specific manner.

Although our understanding of the mechanism of PRL-dependent STAT activation is incomplete, tyrosine phosphorylation of receptor subunits is important for ligand-dependent STAT activation in other cytokine-signaling pathways. STATs interact directly with specific tyrosine residues of receptors, and this is crucial for STAT activation in at least some cases (65, 66). In vitro, the direct phosphorylation of STAT5 by JAK2 to confer specific DNA-binding activity (34) suggests the possibility of alternative mechanisms of STAT activation. Mutation of a single tyrosine residue in the C terminus of the PRLR did not affect JAK2 phosphorylation, but did prevent the induction of ß-casein (33), suggesting that STAT activation by PRL may require STAT-receptor interaction. The sequential activation of STATs 2 and 1 has been proposed in IFN{alpha} signaling (67). The activation of STAT5 by PRL is normal in U3A/PRLR cells, indicating that it is independent of STAT1. However, the sequential activation of STAT5 and then STAT1 by PRL has not been excluded. A more detailed investigation of PRLR, JAK2, and STAT activation using mutant cell lines and dominant-negative STATs could contribute important new information concerning how STATs are activated by PRL.

The promoters of the PRL-induced milk protein genes, including ß-casein and ß-lactoglobulin, and of the IRF-1 gene have GAS elements that can bind to homodimers of STATs 1 or 5 and various heterodimers (14, 39, 44, 47, 68, 69). These observations suggest that STATs 1 and 5 may be involved in the PRL-induced expression of both milk protein and IRF-1 genes. The activation of STAT5 and the induction of milk protein genes have been well characterized (14, 45, 46, 47), but the role of STAT1 in activating these genes remains unclear. The activation of STAT1 by PRL in wild type 2C4/PRLR cells raises the possibility that it may function in this cell type. However, there was no difference in the PRL-dependent induction of ß-lactoglobulin in wild type and STAT1-null cell lines, and IFN{gamma} did not induce ß-lactoglobulin transcription (Fig. 6Go), even though STAT1 was strongly activated (Figs. 2Go and 3Go), arguing against a requirement for STAT1 in the activation of this gene. We cannot exclude the possibility that STAT1 functions in activating other PRL-induced genes. However, recent experiments with STAT1 knockout mice reveal that it is required for IFN signaling (54) but not for apparently normal development, consistent with our findings. In contrast, we observed activation of the IRF-1 gene by ribonuclease (RNase) protection in cells treated with IFN{gamma}, but not with PRL, indicating that STAT5 is not involved in regulation of this process (data not shown).

Recent data demonstrate that, in addition to their important roles in coupling many receptors to the STATs, JAKs may also function to couple some receptors to other pathways (23). Furthermore, kinases other than JAKs, such as those intrinsic to some receptors, may be involved in STAT activation (24, 25, 26, 27, 28). Interestingly, PRL-induced phosphorylation of mitogen-activate protein (MAP) kinase (70), ras (71), and raf-1 (72) have been observed. Further investigation is required to determine the role of ras-dependent signaling in the overall response to PRL and the nature of the kinases involved in this pathway.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Lines, Ligands, and Antisera
We used the parental cell lines 2fTGH and 2C4 and the IFN-unresponsive mutant lines U3A (lacking STAT1) (53, 73), U4C (lacking JAK1), and {gamma}2A (lacking JAK2) (74). Recombinant human IFN{gamma} from Genentech (South San Francisco, CA) and ovine PRL (NIDDK-oPRL-20) from the National Hormone and Pituitary Program (Baltimore, MD) were used at final concentrations of 500 IU/ml or 1 µg/ml, respectively. Anti-STAT1 and anti-STAT2 were gifts of James Darnell, Jr. (Rockefeller University, New York, NY), anti-STAT3 and anti-STAT6 were gifts of James Ihle (St. Jude’s, Memphis, TN). Anti-STAT5b (C-17), which does not recognize STAT5a, was from Santa Cruz Biotechnology (Santa Cruz, CA); peroxidase-conjugated goat antimouse serum was from Boehringer Mannheim (Indianapolis, IN); fluorescein-conjugated goat antimouse serum was from DAKO (Carpinteria, CA); antiphosphotyrosine monoclonal antibodies PY20 and 4G10 were from Transduction Laboratories (Lexington, KY) and Upstate Biotechnology (Lake Placid, NY), respectively; and anti-PRLR monoclonal antibodies mAb M110 and mAb A917 and polyclonal antibody S46 (34) were from Jean Djiane (Institut National de la Recherche Agronomique, Jouy-en-Josas, France).

Transfections and Use of the Fluorescence-Activated Cell Scanner (FACScan)
Cells were stably transfected with the rabbit PRLR cDNA pE-R23 (75) by the calcium phosphate method (76). After selection, cloned cells were incubated with both anti-PRLR monoclonal antibodies (2 µg/ml), followed by fluorescein-conjugated goat antimouse antibody. Clones expressing high levels of PRLR, identified by use of the FACScan, were used in all experiments.

For complementation, vectors expressing wild type murine JAK2 (pRK5/mJAK2) and a kinase-negative mutant protein (pRK5/mJAK2 K>E), kindly provided by James Ihle (St. Jude’s, Memphis, TN) (9, 77), were stably transfected into {gamma}2A/PRLR cells. The levels of JAK2 proteins were analyzed by Western blotting. Clones expressing similar levels of wild type or mutant JAK2 were used in all experiments.

Immunoprecipitation and Western Blot Analyses
Cells were serum-starved overnight and then treated with PRL or IFN{gamma} at 37 C for 10–15 min before cell extracts were prepared. PRLR and STAT proteins, immunoprecipitated as described by Schindler et al. (78), were separated by electrophoresis and adsorbed to polyvinyldifluoridine membranes (Stratagene, La Jolla, CA). Tyrosine phosphorylation was analyzed on Western blots, using antiphosphotyrosine antibodies. The bands were visualized by enhanced chemiluminescence, using Renaissance reagents (DuPont, Boston, MA).

EMSA
Whole-cell extracts, prepared as described by Sadowski et al. (79), were assayed with a 32P-labeled oligonucleotide (5'-AGATTTCTAGGAATTCAATCC-3') corresponding to the GAS element of the bovine ß-casein gene (14). For supershift analyses, the extracts were incubated with preimmune or STAT-specific antisera for 30 min on ice before addition of the labeled probes.

CAT Assays
Cloned cell lines expressing PRLR were stably cotransfected with the ß-lactoglobulin promoter/CAT construct pBJ23 (75) and hygromycin resistance plasmids or transiently transfected with pBJ23 by the calcium phosphate method (76). After selection with hygromycin, pooled cells were serum-starved overnight and then treated with PRL or IFN{gamma} at 37 C for 20 h before cell extracts were prepared. CAT assays were performed with [14C]-labeled chloramphenicol (Amersham, Arlington Heights, IL). The acetylated and nonacetylated forms of chloramphenicol were separated by TLC and quantitated by use of a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Values are expressed as percent chloramphenicol conversion and as fold induction calculated from the basal activity.


    ACKNOWLEDGMENTS
 
We thank James Darnell, Jr., for anti-STATs 1 and 2, James Ihle for anti-STATs 3 and 6 and the JAK2 constructs, Jean Djiane for anti-PRLR, and Jean Djiane and John Clark for pBJ23.


    FOOTNOTES
 
Address requests for reprints to: George R. Stark, Department of Molecular Biology, Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195.

This work was supported by NIH Grant P01 CA-62220.

Received for publication February 26, 1997. Accepted for publication April 7, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Darnell Jr JE, Kerr IM, Stark GR 1994 Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415–1421[Medline]
  2. Ihle JN, Kerr IM 1995 Jaks and Stats in signaling by the cytokine receptor superfamily. Trends Genet 11:69–74[CrossRef][Medline]
  3. Schindler C, Darnell Jr JE 1995 Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu Rev Biochem 64:621–651[CrossRef][Medline]
  4. Wilks AF, Harpur AG, Kurban RR, Ralph SJ, Zürcher G, Ziemiecki A 1991 Two novel protein-tyrosine kinases, each with a second phosphotransferase-related catalytic domain, define a new class of protein kinase. Mol Cell Biol 11:2057–2065[Medline]
  5. Harpur AG, Andres A, Ziemiecki A, Aston RR, Wilks AF 1992 JAK2, a third member of the JAK family of protein tyrosine kinases. Oncogene 7:1347–1353[Medline]
  6. Johnston JA, Kawamura M, Kirken RA, Chen YQ, Blake TB, Shibuya K, Ortaldo JR, McVicar DW, O’Shea JJ 1994 Phosphorylation and activation of the Jak-3 Janus kinase in response to interleukin-2. Nature 370:151–153[CrossRef][Medline]
  7. Witthuhn BA, Silvenoinnen O, Miura O, Lai KS, Cwik C, Liu ET, Ihle JN 1994 Involvement of the Jak-3 Janus kinase in signalling by interleukins 2 and 4 in lymphoid and myeloid cells. Nature 370:153–157[CrossRef][Medline]
  8. Firmbach-Kraft I, Byers M, Shows T, Dalla-Favera R, Krolewski JJ 1990 tyk2, prototype of a novel class of non-receptor tyrosine kinase genes. Oncogene 5:1329–1336[Medline]
  9. Silvennoinen O, Witthuhn BA, Quelle FW, Cleveland JL, Yi T, Ihle JN 1993 Structure of the murine Jak2 protein-tyrosine kinase and its role in interleukin 3 signal transduction. Proc Natl Acad Sci USA 90:8429–8433[Abstract/Free Full Text]
  10. Fu XY, Schindler C, Improta T, Aebersold R, Darnell Jr JE 1992 The proteins of ISGF-3, the interferon alpha-induced transcriptional activator, define a gene family involved in signal transduction. Proc Natl Acad Sci USA 89:7840–7843[Abstract]
  11. Schindler C, Fu XY, Improta T, Aebersold R, Darnell Jr JE 1992 Proteins of transcription factor ISGF-3: one gene encodes the 91- and 84-kDa ISGF-3 proteins that are activated by interferon {alpha}. Proc Natl Acad Sci USA 89:7836–7839[Abstract]
  12. Akira S, Nishio Y, Inoue M, Wang XJ, Wei S, Matsusaka T, Yoshida K, Sudo T, Naruto M, Kishimoto T 1994 Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway. Cell 77:63–71[Medline]
  13. Hou J, Schindler U, Henzel WJ, Ho TC, Brasseur M, McKnight SL 1994 An interleukin-4-induced transcription factor: IL-4 Stat. Science 265:1701–1706[Medline]
  14. Wakao H, Gouilleux F, Groner B 1994 Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response. EMBO J 13:2182–2191[Abstract]
  15. Yamamoto K, Quelle FW, Thierfelder WE, Kreider BL, Gilbert DJ, Jenkins NA, Copeland NG, Silvennoinen O, Ihle JN 1994 Stat4, a novel gamma interferon activation site-binding protein expressed in early myeloid differentiation. Mol Cell Biol 14:4342–4349[Abstract]
  16. Zhong Z, Wen Z, Darnell Jr JE 1994 Stat3 and Stat4: members of the family of signal transducers and activators of transcription. Proc Natl Acad Sci USA 91:4806–4810[Abstract]
  17. Binari R, Perrimon N 1994 Stripe-specific regulation of pair-rule genes by hopscotch, a putative Jak family tyrosine kinase in Drosophila. Genes Dev 8:300–312[Abstract]
  18. Luo H, Hanratty WP, Dearolf CR 1995 An amino acid substitution in the Drosophila hopTum-I Jak kinase causes leukemia-like hematopoietic defects. EMBO J 14:1412–1420[Abstract]
  19. Harrison DA, Binari R, Nahreini TS, Gilman M, Perrimon N 1995 Activation of a Drosophila Janus kinase (JAK) causes hematopoietic neoplasia and developmental defects. EMBO J 14:2857–2865[Abstract]
  20. Sweitzer SM, Calvo S, Kraus MH, Finbloom DS, Larner AC 1995 Characterization of a Stat-like DNA binding activity in Drosophila melanogaster. J Biol Chem 270:16510–16513[Abstract/Free Full Text]
  21. Yan R, Small S, Desplan C, Dearolf CR, Darnell Jr JE 1996 Identification of a Stat gene that functions in Drosophila development. Cell 84:421–430[Medline]
  22. Hou XS, Melnick MB, Perrimon N 1996 marelle acts downstream of the Drosophila HOP/JAK kinase and encodes a protein similar to the mammalian STATs. Cell 84:411–419[Medline]
  23. Han Y, Leaman DW, Watling D, Rogers NC, Groner B, Kerr IM, Wood WI, Stark GR 1996 Participation of JAK and STAT proteins in growth hormone-induced signaling. J Biol Chem 271:5947–5952[Abstract/Free Full Text]
  24. Leaman DW, Pisharody S, Flickinger TW, Commane MA, Schlessinger J, Kerr IM, Levy DE, Stark GR 1996 Roles of JAKs in activation of STATs and stimulation of c-fos gene expression by epidermal growth factor. Mol Cell Biol 16:369–375[Abstract]
  25. David M, Petricoin E3, Benjamin C, Pine R, Weber MJ, Larner AC 1995 Requirement for MAP kinase (ERK2) activity in interferon {alpha}- and interferon ß-stimulated gene expression through STAT proteins. Science 269:1721–1723[Medline]
  26. Boulton TG, Zhong Z, Wen Z, Darnell Jr JE, Stahl N, Yancopoulos GD 1995 STAT3 activation by cytokines utilizing gp130 and related transducers involves a secondary modification requiring an H7-sensitive kinase. Proc Natl Acad Sci USA 92:6915–6919[Abstract]
  27. Yu CL, Meyer DJ, Campbell GS, Larner AC, Carter-Su C, Schwartz J, Jove R 1995 Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein. Science 269:81–83[Medline]
  28. Danial NN, Pernis A, Rothman PB 1995 Jak-STAT signaling induced by the v-abl oncogene. Science 269:1875–1877[Medline]
  29. Kelly PA, Djiane J, Postel-Vinay MC, Edery M 1991 The prolactin/growth hormone receptor family. Endocr Rev 12:235–251[Abstract]
  30. Hooghe R, Delhase M, Vergani P, Malur A, Hooghe-Peters EL 1993 Growth hormone and prolactin are paracrine growth and differentiation factors in the haemopoietic system. Immunol Today 14:212–214[CrossRef][Medline]
  31. Boutin JM, Jolicoeur C, Okamura H, Gagnon J, Edery M, Shirota M, Banville D, Dusanter-Fourt I, Djiane J, Kelly PA 1988 Cloning and expression of the rat prolactin receptor, a member of the growth hormone/prolactin receptor gene family. Cell 53:69–77[Medline]
  32. Kishimoto T, Taga T, Akira S 1994 Cytokine signal transduction. Cell 76:253–262[Medline]
  33. Lebrun JJ, Ali S, Goffin V, Ullrich A, Kelly PA 1995 A single phosphotyrosine residue of the prolactin receptor is responsible for activation of gene transcription. Proc Natl Acad Sci USA 92:4031–4035[Abstract/Free Full Text]
  34. Waters MJ, Daniel N, Bignon C, Djiane J 1995 The rabbit mammary gland prolactin receptor is tyrosine-phosphorylated in response to prolactin in vivo and in vitro. J Biol Chem 270:5136–5143[Abstract/Free Full Text]
  35. Dusanter-Fourt I, Muller O, Ziemiecki A, Mayeux P, Drucker B, Djiane J, Wilks A, Harpur AG, Fischer S, Gisselbrecht S 1994 Identification of JAK protein tyrosine kinases as signaling molecules for prolactin. Functional analysis of prolactin receptor and prolactin-erythropoietin receptor chimera expressed in lymphoid cells. EMBO J 13:2583–2591[Abstract]
  36. Rui H, Kirken RA, Farrar WL 1994 Activation of receptor-associated tyrosine kinase JAK2 by prolactin. J Biol Chem 269:5364–5368[Abstract/Free Full Text]
  37. Campbell GS, Argetsinger LS, Ihle JN, Kelly PA, Rillema JA, Carter-Su C 1994 Activation of JAK2 tyrosine kinase by prolactin receptors in Nb2 cells and mouse mammary gland explants. Proc Natl Acad Sci USA 91:5232–5236[Abstract]
  38. Lebrun JJ, Ali S, Sofer L, Ullrich A, Kelly PA 1994 Prolactin-induced proliferation of Nb2 cells involves tyrosine phosphorylaton of the prolactin receptor and its associated tyrosine kinase JAK2. J Biol Chem 269:14021–14026[Abstract/Free Full Text]
  39. Gilmour KC, Reich NC 1994 Receptor to nucleus signaling by prolactin and interleukin 2 via activation of latent DNA-binding factors. Proc Natl Acad Sci USA 91:6850–6854[Abstract]
  40. David M, Petricoin II EFI, Igarashi KI, Feldman GM, Finbloom DS, Larner AC 1994 Prolactin activates the interferon-regulated p91 transcription factor and the Jak2 kinase by tyrosine phosphorylation. Proc Natl Acad Sci USA 91:7174–7178[Abstract]
  41. Tourkine N, Schindler C, Larose M, Houdebine LM 1995 Activation of STAT factors by prolactin, interferon-gamma, growth hormones, and a tyrosine phosphatase inhibitor in rabbit primary mammary epithelial cells. J Biol Chem 270:20952–20961[Abstract/Free Full Text]
  42. Lai CF, Ripperger J, Morella KK, Wang Y, Gearing DP, Horseman ND, Campos SP, Fey GH, Baumann H 1995 STAT3 and STAT5B are targets of two different signal pathways activated by hematopoietin receptors and control transcription via separate cytokine response elements. J Biol Chem 270:23254–23257[Abstract/Free Full Text]
  43. Gouilleux F, Pallard C, Dusanter-Fourt I, Wakao H, Haldosen LA, Norstedt G, Levy D, Groner B 1995 Prolactin, growth hormone, erythropoietin and granulocyte-macrophage colony stimulating factor induce MGF-Stat5 DNA binding activity. EMBO J 14:2005–2013[Abstract]
  44. Pallard C, Gouilleux F, Charon M, Groner B, Gisselbrecht S, Dusanter-Fourt I 1995 Interleukin-3, erythropoietin, and prolactin activate a STAT5-like factor in lymphoid cells. J Biol Chem 270:15942–15945[Abstract/Free Full Text]
  45. Gouilleux F, Wakao H, Mundt M, Groner B 1994 Prolactin induces phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA binding and induction of transcription. EMBO J 13:4361–4369[Abstract]
  46. Djiane J, Daniel N, Bignon C, Paly J, Waters M, Vacher P, Dufy B 1994 Prolactin receptor and signal transduction to milk protein genes. Proc Soc Exp Biol Med 206:299–303[Abstract]
  47. Groner B, Gouilleux F 1995 Prolactin-mediated gene activation in mammary epithelial cells. Curr Opin Genet Dev 5:587–594[CrossRef][Medline]
  48. Yu-Lee LY, Hrachovy JA, Stevens AM, Schwarz LA 1990 Interferon-regulatory factor 1 is an immediate-early gene under transcriptional regulation by prolactin in Nb2 T cells. Mol Cell Biol 10:3087–3094[Medline]
  49. O’Neal KD, Yu-Lee LY 1994 Differential signal transduction of the short, Nb2, and long prolactin receptors. Activation of interferon regulatory factor-1 and cell proliferation. J Biol Chem 269:26076–26082[Abstract/Free Full Text]
  50. Stevens AM, Yu-Lee LY 1992 The transcription factor interferon regulatory factor-1 is expressed during both early G1 and the G1/S transition in the prolactin-induced lymphocyte cell cycle. Mol Endocrinol 6:2236–2243[Abstract]
  51. Guschin D, Rogers N, Briscoe J, Witthuhn B, Watling D, Horn F, Pellegrini S, Yasukawa K, Heinrich P, Stark GR, Ihle JN, Kerr IM 1995 A major role for the protein tyrosine kinase JAK1 in the JAK/STAT signal transduction pathway in response to interleukin-6. EMBO J 14:1421–1429[Abstract]
  52. Vignais M-L, Sadowski HB, Watling D, Rogers NC, Gilman M 1996 Platelet-derived growth factor induces phosphorylation of multiple JAK family kinases and STAT proteins. Mol Cell Biol 16:1759–1769[Abstract]
  53. Müller M, Laxton C, Briscoe J, Schindler C, Improta T, Darnell Jr JE, Stark GR, Kerr IM 1993 Complementation of a mutant cell line: central role of the 91 kDa polypeptide of ISGF3 in the interferon-alpha and -gamma signal transduction pathways. EMBO J 12:4221–4228[Abstract]
  54. Meraz MA, White JM, Sheehan KCF, Bach EA, Rodig SJ, Dighe AS, Kaplan DH, Riley JK, Greenlund AC, Campbell D, Carver-Moore K, DuBois RN, Clark R, Aguet M, Schreiber RD 1996 Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell 84:431–442[Medline]
  55. Lebrun J-J, Ali S, Ullrich A, Kelly PA 1995 Proline-rich sequence-mediated Jak2 association to the prolactin receptor is required but not sufficient for signal transduction. J Biol Chem 270:10664–10670[Abstract/Free Full Text]
  56. Ali S, Pellegrini I, Kelly PA 1991 A prolactin-dependent immune cell line (Nb2) expresses a mutant form of prolactin receptor. J Biol Chem 266:20110–20117[Abstract/Free Full Text]
  57. Stahl N, Boulton TG, Farruggella T, Ip NY, Davis S, Witthuhn BA, Quelle FW, Silvennoinen O, Barbieri G, Pellegrini S, Ihle JN, Yancopoulos GD 1994 Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 ß receptor components. Science 263:92–95[Medline]
  58. Azam M, Erdjument-Bromage H, Kreider BL, Xia M, Quelle F, Basu R, Saris C, Tempst P, Ihle JN, Schindler C 1995 Interleukin-3 signals through multiple isoforms of Stat5. EMBO J 14:1402–1411[Abstract]
  59. Mui AL-F, Wakao H, O’Farrell A-M, Harada N, Miyajima A 1995 Interleukin-3, granulocyte-macrophage colony stimulating factor and interleukin-5 transduce signals through two STAT5 homologs. EMBO J 14:1166–1175[Abstract]
  60. Liu X, Robinson GW, Gouilleux F, Groner B, Hennighausen L 1995 Cloning and expression of Stat5 and an additional homologue (Stat5b) involved in prolactin signal transduction in mouse mammary tissue. Proc Natl Acad Sci USA 92:8831–8835[Abstract]
  61. Lin J-X, Mietz J, Modi WS, John S, Leonard WJ 1996 Cloning of human Stat5B. J Biol Chem 271:10738–10744[Abstract/Free Full Text]
  62. Quelle FW, Wang D, Nosaka T, Thierfelder WE, Stravopodis D, Weinstein Y, Ihle JN 1996 Erythropoietin induces activation of Stat5 through association with specific tyrosines on the receptor that are not required for a mitogenic response. Mol Cell Biol 16:1621–1631
  63. DaSilva L, Rui H, Erwin RA, Howard OMZ, Kirken RA, Malabarba MG, Hackett RH, Larner AC, Farrar WL 1996 Prolactin recruits STAT1, STAT3 and STAT5 independent of conserved receptor tyrosines TYR402, TYR479, TYR515 and TYR580. Mol Cell Endocrinol 117:131–140[CrossRef][Medline]
  64. Ruff-Jamison S, Chen K, Cohen S 1995 Epidermal growth factor induces the tyrosine phosphorylation and nuclear translocation of Stat5 in mouse liver. Proc Natl Acad Sci USA 92:4215–4218[Abstract]
  65. Greenlund AC, Farrar MA, Viviano BL, Schreiber RD 1994 Ligand-induced IFNgamma receptor tyrosine phosphorylation couples the receptor to its signal transduction system (p91). EMBO J 13:1591–1600[Abstract]
  66. Stahl N, Farruggella TJ, Boulton TG, Zhong Z, Darnell Jr JE, Yancopoulos GD 1995 Choice of STATs and other substrates specified by modular tyrosine-based motifs in cytokine receptors. Science 267:1349–1353[Medline]
  67. Leung S, Qureshi SA, Kerr IM, Darnell Jr JE, Stark GR 1995 Role of STAT2 in the alpha interferon signaling pathway. Mol Cell Biol 15:1312–1317[Abstract]
  68. Schmitt-Ney M, Happ B, Hofer P, Hynes NE, Groner B 1992 Mammary gland-specific nuclear factor activity is positively regulated by lactogenic hormones and negatively by milk stasis. Mol Endocrinol 6:1988–1997[Abstract]
  69. Sims SH, Cha Y, Romine MF, Gao P-Q, Gottlieb K, Deisseroth AB 1993 A novel interferon-inducible domain: structural and functional analysis of the human interferon regulatory factor 1 gene promoter. Mol Cell Biol 13:690–702[Abstract]
  70. Carey GB, Liberti JP 1995 Stimulation of receptor-associated kinase, tyrosine kinase, and MAP kinase is required for prolactin-mediated macromolecular biosynthesis and mitogenesis in Nb2 lymphoma. Arch Biochem Biophys 316:179–189[CrossRef][Medline]
  71. Erwin RA, Kirken RA, Malabarba MG, Farrar WL, Rui H 1995 Prolactin activates Ras via signaling proteins SHC, growth factor reception bound 2, and son of sevenless. Endocrinology 136:3512–3518[Abstract]
  72. Clevenger CV, Torigoe T, Reed JC 1994 Prolactin induces rapid phosphorylation and activation of prolactin receptor-associated RAF-1 kinase in a T-cell line. J Biol Chem 269:5559–5565[Abstract/Free Full Text]
  73. John J, McKendry R, Pellegrini S, Flavell D, Kerr IM, Stark GR 1991 Isolation and characterization of a new mutant human cell line unresponsive to alpha and beta interferons. Mol Cell Biol 11:4189–4195[Medline]
  74. Watling D, Guschin D, Muller M, Silvennoinen O, Witthuhn BA, Quelle FW, Rogers NC, Schindler C, Stark GR, Ihle JN 1993 Complementation by the protein tyrosine kinase JAK2 of a mutant cell line defective in the interferon-gamma signal transduction pathway. Nature 366:166–170[CrossRef][Medline]
  75. Lesueur L, Edery M, Paly J, Clark J, Kelly PA, Djiane J 1990 Prolactin stimulates milk protein promoter in CHO cells cotransfected with prolactin receptor cDNA. Mol Cell Endocrinol 71:R7–R12
  76. Kingston R 1987 Transfection of DNA into eukaryotic cells. In: Ausubel FA, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current Protocols in Molecular Biology. Greene and Wiley Interscience, New York, pp 911–914
  77. Briscoe J, Rogers NC, Witthuhn BA, Watling D, Harpur AG, Wilks AF, Stark GR, Ihle JN, Kerr IM 1996 Kinase-negative mutants of JAK1 can sustain interferon-gamma-inducible gene expression but not an antiviral state. EMBO J 15:799–809[Abstract]
  78. Schindler C, Shuai K, Prezioso VR, Darnell Jr JE 1992 Interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor. Science 257:809–813[Medline]
  79. Sadowski HB, Gilman MZ 1993 Cell-free activation of a DNA-binding protein by epidermal growth factor. Nature 362:79–83[CrossRef][Medline]