©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning of Human Stat5B
RECONSTITUTION OF INTERLEUKIN-2-INDUCED Stat5A AND Stat5B DNA BINDING ACTIVITY IN COS-7 CELLS (*)

(Received for publication, December 18, 1995; and in revised form, February 2, 1996)

Jian-Xin Lin (1) Judy Mietz (1) William S. Modi (2) Susan John (1) Warren J. Leonard (1)(§)

From the  (1)Laboratory of Molecular Immunology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892 and the (2)Biological Carcinogenesis and Development Program, Science Applications International Corp., NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have isolated a second human Stat5 cDNA, Stat5B, and demonstrated that the genes encoding both Stat5A and Stat5B are located at chromosome 17q11.2. Both genes were constitutively transcribed in peripheral blood lymphocytes. By using specific antisera, we demonstrated that both Stat5A and Stat5B are activated by interleukin-2 (IL-2) in peripheral blood lymphocytes, natural killer-like YT leukemia cells, and human T cell lymphotropic virus type I-transformed MT-2 T cells. In COS-7 cells, which constitutively express the Janus family tyrosine kinase Jak1, reconstitution of IL-2-induced Stat5A and Stat5B DNA binding activities was dependent on the coexpression of Jak3 along with the IL-2 receptor beta chain and the common cytokine receptor -chain. This IL-2-induced Stat5 activation was dependent on the presence of either of two tyrosines (Tyr-392 or Tyr-510) in the IL-2 receptor beta chain, indicating that either of these two tyrosines can serve as a docking site. Moreover, we demonstrated that human Stat5 activation is also dependent on Tyr-694 in Stat5A and Tyr-699 in Stat5B, indicating that these tyrosines are required for dimerization. The COS-7 reconstitution system described herein provides a valuable assay for further elucidation of the IL-2-activated JAK-STAT pathway.


INTRODUCTION

The interaction of interleukin-2 (IL-2) (^1)with IL-2 receptors (IL-2R) on lymphocytes, natural killer cells, and monocytes induces pleiotropic biological effects on the immune system. Functional IL-2Rs contain IL-2Rbeta and the common cytokine receptor -chain ((c)) (1, 2, 3) , two members of the cytokine receptor superfamily(4) . Intermediate affinity receptors contain only IL-2Rbeta and (c), whereas high affinity receptors additionally contain IL-2Ralpha; both of these forms of IL-2 receptors are capable of transducing IL-2 signals(2, 3) . IL-2Rbeta is also a component of the IL-15 receptor(5, 6) , while (c) is shared by the receptors for IL-2(1) , IL-4(7, 8) , IL-7(9, 10) , IL-9(11, 12) , and IL-15(5) . In humans, mutation of the (c) gene can result in X-linked severe combined immunodeficiency(13, 14, 15) .

IL-2 stimulation of lymphocytes rapidly activates the Janus family tyrosine kinases Jak1 and Jak3(11, 16, 17) . Jak1 associates with IL-2Rbeta, and Jak3 primarily associates with (c)(11, 18, 19) . JAK family kinases play critical signaling roles for interferons and for many other cytokines by activating STAT (signal transducers and activators of transcription) proteins that then dimerize, rapidly translocate into the nucleus, and modulate gene expression(20, 21) . At least six different human STAT genes have been identified (20, 21) . They encode an even greater number of protein products due to alternative mRNA splicing. Many cytokines are known to activate more than a single STAT protein, allowing for additional complexity due to the formation of both homo- and heterodimers between different STAT proteins. For example, IL-2 rapidly activates Stat5 in freshly isolated peripheral blood lymphocytes (PBL) and both Stat3 and Stat5 in PBL preactivated for 72 h with phytohemagglutinin (PHA) (22) and in natural killer-like YT leukemia cells(22, 23) .

We now report the cloning of a second human Stat5 cDNA (Stat5B) and the chromosomal localization of both human Stat5A and Stat5B. We also provide evidence that both Stat5A and Stat5B are activated in normal PBL by IL-2 in vivo. Using a transient transfection system in COS-7 cells, we have reconstituted IL-2-induced activation of both Stat5A and Stat5B, shown that either Tyr-392 or Tyr-510 of IL-2Rbeta is required for IL-2-dependent Stat5 activation, and identified a tyrosine residue in each human Stat5 protein that is essential for its activation by Jak1 and Jak3.


EXPERIMENTAL PROCEDURES

Cell Lines

COS-7 cells (American Type Culture Collection) were cultured in Dulbecco's modified essential medium containing 10% fetal bovine serum, 2 mM glutamine, and 100 units/ml penicillin and streptomycin. Human T cell lymphotropic virus type I (HTLVI)-transformed MT-2 T cells and natural killer-like YT cells (24) and their subclone (YT-1) were grown in RPMI 1640 medium supplemented as described for COS-7 cells.

Isolation of Human Stat5 cDNAs and Chromosomal Localization

Primers P1 (nucleotides 1572-1597, 5`-GTTTGAGTCTCAATTCAGTGTTGGCA-3`) and P2 (nucleotides 2107 to 2084, 5`-AGTCGCTAAAGCGCAACAAGAAGG-3`) of ovine Stat5 (25) were used to amplify human Stat5 by PCR; the primers were specific for Stat5 based on their inability to amplify Stat1, Stat2, Stat3, and Stat4. PCR was performed for 30 cycles (denaturing at 94 °C for 30 s, annealing at 59 °C for 30 s, and extending at 72 °C for 1 min) using template DNA from a YT-1 cDNA library in the eukaryotic expression vector pME18S. The resulting 535-base pair fragment was radiolabeled and used to screen the YT-1 cDNA library. Complementary DNAs containing the entire Stat5B and partial Stat5A coding regions were identified. A cDNA encoding the entire Stat5A coding region was obtained by screening a YT-ZAPII cDNA library (26) with a probe generated by reverse transcription-PCR (Perkin-Elmer) of normal donor PBL RNA by using human Stat5A-specific primers P3 (nucleotides 1973-1996, 5`-GAGTCTCAGTTCAGTGTTGGCAGC-3`) and P4 (nucleotides 2504 to 2479, 5`-AGTCACTAAAGCGCAACAAGAAGGTC-3`). For both Stat5A and Stat5B cDNAs, both strands were sequenced (Rapid-Denature plasmid sequencing kit, Amersham-United Biochemical Inc.).

The NaeI-XhoI fragment of human Stat5A was subcloned between the EcoRV and XhoI sites of pSX, a eukaryotic expression vector; the Stat5B cDNA was subcloned between the EcoRI and NotI sites of pSX. The resulting plasmids are denoted pSXStat5A and pSXStat5B, respectively. For chromosomal localization, fluorescent in situ hybridization was performed using human leukocytes from normal donors as described (27) with pSXStat5A and pSXStat5B as probes.

Northern Blot Analysis

RNA was isolated using RNAzol B (Tel-Test, Inc., Friendswood, TX). Hybridization probes were made by PCR using Stat5A primers P5 (nucleotides 3032-3056, 5`-GAATCCCACGCTTCTCTTTGGAAAC-3`) and P6 (nucleotides 3614 to 3590, 5`-CAGAGAGTCTGGAGTCCACGTTCAC-3`) and Stat5B primers P7 (nucleotides 2484-2505, 5`-CAGTGGATCCCGCACGCACAAT-3`) and P8 (nucleotides 2601 to 2580, 5`-TCAAAAGAGAAGCGATTCATGG-3`).

Plasmids, in Vitro Transcription/Translation, and Site-directed Mutagenesis

Human IL-2Rbeta, (c), and Jak3 cDNAs were subcloned in pME18S(11, 28) ; murine Jak1 was subcloned in pMLCMV. The IL-2Rbeta tyrosine mutants were prepared as described(29) . A full-length phagemid Stat5A cDNA in pBluescript is denoted as pBS-Stat5A. The SmaI-XhoI fragment of Stat5B was subcloned between the EcoRV and XhoI sites of pBluescript SK vector to generate pBS-Stat5B. pBS-Stat5A and pBS-Stat5B DNAs were used for in vitro transcription/translation using the TNT T3-coupled reticulocyte lysate system (Promega) and [S]methionine. To mutate Tyr-694 of Stat5A and Tyr-699 of Stat5B to phenylalanines, the primers 5`-GCTAAAGCTGTTGATGGATTTGTGAAACCACAGATC-3` and 5`-GCTAAAGCTGTTGATGGATTCGTGAAGCCACAGATC-3` were used, respectively (mutated nucleotides are underlined), in conjunction with the Morph site-specific plasmid DNA mutagenesis kit (5 Prime 3 Prime, Inc., Boulder, CO).

Peptides and Antisera

Stat5A (residues 778-794, NH(2)-CRLSPPAGLFTSARGSLS-COOH) and Stat5B (residues 774-787, NH(2)-CGRPMDSQWIPHAQS-COOH) peptides (synthesized on an Applied Biosystems 431A peptide synthesizer; the NH(2)-terminal cysteines were added) were coupled to Imject maleimide-activated keyhole limpet hemocyanin (Pierce), and rabbit antisera were produced (Duncroft Inc., Lovettsville, VA). Anti-phosphotyrosine monoclonal antibody (4G10) was from Upstate Biotechnology, Inc. (Lake Placid, NY).

Nuclear Extracts, Electrophoretic Mobility Shift Assays, and DNA Affinity Purification

COS-7 cells were transiently transfected (LipofectAMINE, Life Technologies, Inc.) in 100-mm dishes using 2 µg of each plasmid plus sufficient pSX to yield a total of 14 µg. Two days later, cells were either not treated or treated with 1 nM IL-2 for 20 min; nuclear extracts were prepared(30) ; and electrophoretic mobility shift assays were performed as described (22) using 10 µg of the nuclear extracts and 15,000 cpm of P-labeled GAS motif (5`-AGATTTCTAGGAATTC-3`) from the beta-casein promoter to which a HindIII site was added to the 5`-end and a BamHI site to the 3`-end. For DNA affinity purification(22) , we used double-stranded biotinylated oligonucleotides containing a trimer of beta-casein motifs.


RESULTS

Isolation of Two Closely Related Human Stat5 cDNAs Whose Genes Are Localized on Chromosome 17

Stat5 was originally isolated as the prolactin-induced ovine mammary gland transcription factor(25) . It is now also known to be activated by IL-2(22, 23, 31, 32, 33) , IL-3(34, 35) , granulocyte-macrophage colony-stimulating factor(34, 36) , IL-7 (22) , IL-15(22) , erythropoietin(36) , growth hormone(36) , and thrombopoietin(37) . Corresponding to the single ovine Stat5 cDNA, a single human Stat5 was identified as an IL-2-induced STAT protein (23) , but two closely related murine Stat5 proteins (Stat5A and Stat5B) were found to be induced by IL-3 and granulocyte-macrophage colony-stimulating factor(34, 35) . In analyzing human Stat5 cDNAs, we have now isolated human homologues for both murine Stat5A and Stat5B. The coding regions are 91 and 84% identical at the nucleotide level and 93 and 87% identical at the amino acid level to ovine Stat5 (Fig. 1). Human Stat5A is identical to the known human Stat5 cDNA (23) and 91% identical to Stat5B at the amino acid level. The putative DNA-binding(38, 39) , SH3(25) , and SH2 (25) domains of Stat5A and Stat5B are highly conserved, whereas the COOH-terminal 21 and 9 amino acids of Stat5A and Stat5B, respectively, are unique (Fig. 1). We also isolated a cDNA denoted Stat5B-2 that was identical to Stat5B except for a deletion of 93 nucleotides encoding 31 amino acids in the DNA-binding domain and that was presumably generated by alternative splicing. The genes for both Stat5A and Stat5B colocalize at human chromosome 17q11.2 (Fig. 2), a region syntenic to the distal region of murine chromosome 11, where the genes for both murine Stat5A and Stat5B were colocalized(40) ; in contrast, the genes encoding Stat1 and Stat4 have been localized on murine chromosome 1 in a region syntenic to human chromosome 2(41) .


Figure 1: Alignment of amino acid sequences of human Stat5A and Stat5B, murine Stat5A and Stat5B, and ovine Stat5. Residue numbers are on the right. Dots indicate identical nucleotides; hyphens are gaps introduced to optimize alignment. The putative DNA-binding(38, 39) , SH3(25) , and SH2 domains (25) and the phosphorylated tyrosines (23, 25, 34) are boxed. The sequences for Stat5 proteins are from the following sources: Stat5A ( (23) and this study; GenBank accession numbers L41142 and U43185), Stat5B (this study; GenBank accession number U47686), murine Stat5A ((34) ; GenBank accession number Z48538), murine Stat5B ((34) ; GenBank accession number Z48539), and ovine Stat5 ((25) ; GenBank accession number X78428). Hu, human; Mu, murine; MGF, mammary gland transcription factor.




Figure 2: Human Stat5A and Stat5B genes map to chromosome 17. Shown are metaphase cells following fluorescent in situ hybridization with human Stat5A (A) and Stat5B (B) cDNAs. 35 of 72 cells examined for Stat5A and 23 of 58 cells examined for Stat5B exhibited paired hybridization signals at 17q11.2 (arrows), and an additional seven (for Stat5A) and eight (for Stat5B) cells showed one hybridization signal at the same locus. No significant background was noted at any other chromosomal location. Chromosomes were identified using Quinacrine Fluorescence Hoechst (QFH) banding by simultaneous Hoechst 33258 staining (not shown).



Human Stat5A and Stat5B Are Transcribed as Different mRNAs

At least two major Stat5 transcripts of 6 and 4 kb have been identified in a study using a human Stat5A cDNA probe (23) that would cross-hybridize with Stat5B based on their sequence similarities. Using probes corresponding to unique 3`-untranslated regions of Stat5A and Stat5B, we have found that major transcripts of 3.8 kb for Stat5A (Fig. 3, lane 1) and 5 kb for Stat5B (lane 2) are transcribed in human PBL. mRNA encoding Stat5B-2 could not be detected even by PCR in normal human PBL (data not shown), suggesting that this form is unlikely to be abundant enough to be physiologically important as a putative DNA binding-defective dominant negative form of Stat5.


Figure 3: Stat5A and Stat5B transcripts differ in size. Total RNA from fresh PBL was separated on a 1% formaldehyde-agarose gel, blotted onto a nylon membrane, and hybridized sequentially with P-labeled probes specific for human Stat5A (lane 1) and Stat5B (lane 2). Although the major Stat5A mRNA detected by Northern blotting is 3.8 kb, we have isolated one Stat5A cDNA of 5.5 kb in a YT cDNA library, indicating that longer transcripts also exist. It is therefore conceivable that alternative polyadenylation may explain this finding.



IL-2 Activates Both Stat5A and Stat5B in Vivo

Although IL-2 is known to activate Stat5 binding activity in vivo, it has been unclear whether Stat5A or Stat5B or both were activated. By immunoprecipitation with antisera specific for Stat5A and Stat5B (Fig. 4A) followed by Western blotting with anti-phosphotyrosine antibodies (Fig. 4B, upper panels), we found that both Stat5A (lanes 2 and 4) and Stat5B (lanes 6 and 8) were activated by IL-2 in PBL and YT cells. The major Stat5A band was 95 kDa, while a Stat5B doublet of 90 and 92 kDa was identified (Fig. 4B, lower panels). Since the 92-kDa band of Stat5B was predominant in anti-phosphotyrosine blotting and was present only in the lysates from IL-2-activated cells (Fig. 4B, lanes 6 and 8 versus lanes 5 and 7; compare upper and lower panels), it likely represents a hyperphosphorylated form of the 90-kDa band. DNA affinity purification studies using the beta-casein GAS oligonucleotide revealed that Stat5A and both forms of Stat5B were present in IL-2-induced complexes formed from nuclear extracts from fresh PBL, PBL preactivated with PHA, human T cell lymphotropic virus type I-transformed MT-2 cells, and YT cells (Fig. 4, C and D).


Figure 4: Both Stat5A and Stat5B are activated by IL-2 and can bind DNA. A, specificity of Stat5A and Stat5B antisera. [S]Methionine-labeled rabbit reticulocyte lysates not programed (lanes 1 and 4) or programed with human Stat5A (lanes 2 and 5) or Stat5B (lanes 3 and 6) were immunoprecipitated (IP) by R1216 anti-Stat5A (lanes 1-3) or R1219 anti-Stat5B (lanes 4-6) antisera and analyzed on 8% SDS gels (Novex, San Diego, CA). B, Stat5A and Stat5B are activated by IL-2 in PBL and YT cells. PBL preactivated with PHA for 3 days and rested overnight (lanes 1, 2, 5, and 6) and YT cells (lanes 3, 4, 7, and 8) were not treated (lanes 1, 3, 5, and 7) or were treated with IL-2 (lanes 2, 4, 6, and 8). Cellular lysates were immunoprecipitated with anti-Stat5A (lanes 1-4) or anti-Stat5B (lanes 5-8); subjected to Western blotting with 4G10 (upper panels), anti-Stat5A (lower left panel), or anti-Stat5B (lower right panel); and developed by ECL. C and D, activation by IL-2 of Stat5A and Stat5B in nuclear extracts from fresh PBL (lanes 1), PBL preactivated by PHA (lanes 2), MT-2 cells (lanes 3), and YT cells (lanes 4). STAT proteins were purified by GAS motif DNA affinity columns and subjected to Western blotting with anti-Stat5A (C) or anti-Stat5B (D). In each panel, molecular masses (in kilodaltons) are indicated on the left, based on the mobilities of Seeblue markers (Novex).



Reconstitution of IL-2-induced Stat5 Activation in COS-7 Cells

We next investigated if IL-2-induced Stat5 DNA binding activity could be reconstituted in COS-7 cells, which have endogenous Jak1, but do not express IL-2Rbeta, (c), or Jak3 ( (29) and data not shown). We transfected COS-7 cells with various combinations of expression vectors and evaluated the ability of IL-2 to activate Stat5 DNA binding activity by electrophoretic mobility shift assays using a GAS motif from the beta-casein promoter, which is known to bind to Stat5 ( Fig. 5and results summarized in Table 1)(15) . No DNA binding to the beta-casein GAS was detected in COS-7 cells that were mock-transfected or in cells transfected with IL-2Rbeta and (c) cDNAs or with IL-2Rbeta, (c), and Jak3 cDNAs if STAT cDNAs were omitted from the transfection (Fig. 5A, lanes 1-4, 7, and 8). When IL-2Rbeta, (c), and Jak1 cDNAs were cotransfected, a basal DNA binding activity was detected whose level was not significantly affected by IL-2 treatment (lanes 5 and 6). Based on antibody reactivity, this complex contains Stat1 (data not shown). These results confirmed that there was no detectable endogenous Stat5 binding activity in COS-7 cells, indicating that they would be useful for investigating the requirement for Stat5 activation in response to IL-2. Cotransfection of the Jak1 cDNA with Stat5A, Stat5B, IL-2Rbeta, and (c) cDNAs yielded high levels of a new complex with slower mobility (lane 9) that was supershifted by anti-Stat5 antiserum (data not shown); the new complex was not induced by IL-2 (lane 10). A similar high level of IL-2-independent Stat5 binding activity was observed in cells transfected with both Jak1 and Jak3, Stat5A, Stat5B, IL-2Rbeta, and (c) cDNAs (data not shown). Thus, in these cells, overexpression of Jak1 alone or in combination with Jak3 resulted in constitutive high level Stat5 binding activity. However, when Jak3 was the only JAK family kinase in the transfection, there was a lower level of basal Stat5 binding activity (lane 11 versus lane 9); and this binding activity was potently induced by IL-2 (lanes 12 versus lane 11). Since COS-7 cells express low levels of endogenous Jak1, we hypothesize that this successful reconstitution of IL-2-induced Stat5 DNA binding activity may be mediated by the presence of low levels of endogenous Jak1 and the transfected Jak3 acting together in concert. As expected, no DNA binding activity was detected if both Jak1 and Jak3 cDNAs were omitted from the transfection (lanes 13 and 14), indicating that the low levels of endogenous Jak1 were not sufficient to mediate the activation of exogenously provided Stat5.


Figure 5: Reconstitution of Stat5 binding activity in COS-7 cells: demonstration that Tyr-392 and Tyr-510 of IL-2Rbeta, Tyr-694 of Stat5A, and Tyr-699 of Stat5B are required for IL-2-induced Stat5 activation. A, COS-7 cells were transfected with pSX (lanes 1 and 2); IL-2Rbeta and (c) cDNAs (lanes 3 and 4); IL-2Rbeta, (c), and Jak1 cDNAs (lanes 5 and 6); IL-2Rbeta, (c), and Jak3 cDNAs (lanes 7 and 8); IL-2Rbeta, (c), Jak1, Stat5A, and Stat5B cDNAs (lanes 9 and 10); IL-2Rbeta, (c), Jak3, Stat5A, and Stat5B cDNAs (lanes 11 and 12); and IL-2Rbeta, (c), Stat5A, and Stat5B cDNAs (lanes 13 and 14). Two days later, cells were either not treated (lanes 1, 3, 5, 7, 9, 11, and 13) or treated with IL-2 (lanes 2, 4, 6, 8, 10, 12, and 14), and nuclear extracts were prepared. The specific complexes formed with endogenous Stat1 and transfected Stat5 proteins are indicated. Although some COS-7 cells have higher levels of Stat1 than others, the pattern for IL-2-dependent Stat5 activation was consistent (data not shown). B, Stat5A (lanes 1 and 2), Stat5B (lanes 3 and 4), or both Stat5A and Stat5B (lanes 5 and 6) cDNAs were cotransfected into COS-7 cells with IL-2Rbeta, (c), and Jak3 cDNAs. Two days later, nuclear extracts were prepared from cells not treated (lanes 1, 3, and 5) or treated with IL-2 (lanes 2, 4, and 6). Similar levels of Stat5B expression (lanes 3 and 4 versus lanes 5 and 6) were confirmed by Western blotting (data not shown). C, wild-type IL-2Rbeta (WT; lanes 1 and 2) or IL-2Rbeta mutated at Tyr-338, Tyr-355, Tyr-358, and Tyr-361 (IL-2Rbeta FFFFYY; lanes 3 and 4), or Tyr-392 (IL-2Rbeta YYYYFY; lanes 5 and 6), or Tyr-510 (IL-2Rbeta YYYYYF; lanes 7 and 8), or both Tyr-392 and Tyr-510 (IL-2Rbeta YYYYFF; lanes 9 and 10) was cotransfected with (c), Jak3, Stat5A, and Stat5B into COS-7 cells. Two days after transfection, nuclear extracts were prepared from cells not treated (lanes 1, 3, 5, 7, and 9) or treated with IL-2 (lanes 2, 4, 6, 8, and 10). D, COS-7 cells were transfected with IL-2Rbeta and Jak1 (lane 1); IL-2Rbeta, Jak1, and Stat5B (lane 2); Stat5B and Jak1 (lane 3); or IL-2Rbeta and Stat5B (lane 4). E, COS-7 cells were transfected with (c) and Jak3 (lane 1); (c), Jak3, and Stat5B (lane 2); Stat5B and Jak3 (lane 3); or (c) and Stat5B (lane 4). F, Stat5A (lanes 1 and 2), Stat5A Y694F (lanes 3 and 4), Stat5B (lanes 5 and 6), and Stat5B Y699F (lanes 7 and 8) were cotransfected into COS-7 cells with IL-2Rbeta, (c), and Jak3 cDNAs. 48 h later, COS-7 cells were not treated (lanes 1, 3, 5, and 7) or treated with IL-2 (lanes 2, 4, 6, and 8), and nuclear extracts were prepared. G, Stat5A (WT; lane 1), Stat5A Y694F (lane 2), Stat5B (WT; lane 3), and Stat5B Y699F (lane 4) cDNAs were cotransfected into COS-7 cells with IL-2Rbeta, (c), and Jak1 cDNAs. For F and G, expression levels of wild-type and mutant Stat5 proteins were similar (data not shown). For D-G, 2 days after transfection, nuclear extracts were prepared, and electrophoretic mobility shift assays were performed using the beta-casein probe.





The above experiments were performed using a combination of Stat5A and Stat5B. We found that either Stat5A or Stat5B alone could also result in DNA binding to the beta-casein probe (Fig. 5B, lanes 1-4), suggesting that both Stat5A and Stat5B homodimers could bind to the probe. The greater binding activity seen with Stat5B compared with Stat5A (lane 4 versus lane 2) was likely due to the greater expression of Stat5B in the transfected COS-7 cells (data not shown). Coexpression of Stat5A and Stat5B consistently resulted in a greater DNA binding activity than the sum of the binding seen with Stat5A and Stat5B alone (lane 6 versus lanes 4 and 2), suggesting that Stat5A-Stat5B heterodimers can also bind DNA.

Both Tyrosines 392 and 510 of IL-2Rbeta Are Required for Stat5 Activation

We have recently shown that tyrosines 392 and 510 of IL-2Rbeta appear to be docking sites for IL-2-induced STAT proteins and that 32D cells transfected with a mutant IL-2Rbeta lacking Tyr-392 and Tyr-510 failed to mediate STAT activation by IL-2(22) . To further evaluate the COS reconstitution system, we determined whether Tyr-392 or Tyr-510 or both were required for Stat5 activation by IL-2 in COS-7 cells. Consistent with our previous findings, mutation of the four proximal tyrosines (Tyr-338, Tyr-355, Tyr-358, and Tyr-361) to phenylalanines (IL-2Rbeta FFFFYY) had no effect on activation of Stat5 proteins in COS-7 cells (Fig. 5C, lane 4 versus lane 2). A double mutation of Tyr-392 and Tyr-510 (IL-2Rbeta YYYYFF) markedly diminished both constitutive and IL-2-induced Stat5 activation (lanes 9 and 10 versus lanes 1 and 2), whereas selective mutation of either Tyr-392 (IL-2Rbeta YYYYFY) or Tyr-510 (IL-2Rbeta YYYYYF) still allowed significant Stat5 activation (lanes 5-8). These data establish that either Tyr-392 or Tyr-510 is required for potent activation of both Stat5A and Stat5B in response to IL-2.

Although Stat5 DNA binding activity was greatly diminished in COS-7 cells transfected with the double mutation of Tyr-392 and Tyr-510 of IL-2Rbeta, it was still detectable (Fig. 5C, lanes 9 and 10). This result appears to be somewhat different from our previous observation that a truncated form of the IL-2Rbeta chain lacking Tyr-392 and Tyr-510 is not able to mediate STAT protein activation by IL-2 in 32D cells(22) . We reasoned that the difference might reflect the higher levels of JAK kinases and/or Stat5 proteins in COS-7 transfectants than in 32D cells. Indeed, either Jak1 or Jak3 can activate Stat5B in the absence of receptor chains, although IL-2Rbeta facilitates Stat5B activation (Fig. 5, D and E). Transfection of COS-7 cells with IL-2Rbeta and Jak1 (without Stat5B) activated endogenous Stat1 (Fig. 5D, lane 1). Although overexpression of Jak1 activated transfected Stat5B (lane 3), coexpression of Jak1 with IL-2Rbeta caused a higher level of activation of Stat5B than that seen with Jak1 alone (lane 2 versus lane 3). In contrast, coexpression of (c) with Jak3 did not augment Stat5B activation more than Jak3 alone (Fig. 5E, lane 2 versus lane 3). This is consistent with the presence of STAT protein docking sites on IL-2Rbeta, but not on (c)(22, 31, 32) . These results demonstrate that overexpression of Stat5 with either Jak1 or Jak3 is sufficient to cause Stat5 activation, suggesting that when the level of expression and/or activities of JAK kinases and/or STAT proteins are disregulated, STAT proteins may directly associate with JAK kinases without requiring receptor docking sites for the activation. However, under physiological conditions, the presence of docking sites on IL-2Rbeta is clearly required for Stat5 activation by IL-2(22, 31, 32) .

Tyr-694 of Stat5A and Tyr-699 of Stat5B Are Necessary for Activation

Cytokine-induced phosphorylation on a conserved tyrosine residue of STAT proteins is essential for their dimerization and acquisition of DNA binding activity(20) . In ovine Stat5, Tyr-694 is phosphorylated in response to prolactin(36) . Since prolactin activates Jak2 (42, 43) and IL-2 instead activates Jak1 and Jak3(11, 18, 19) , we tested whether phosphorylation of the homologous tyrosines in human Stat5 (Tyr-694 for Stat5A and Tyr-699 for Stat5B) was required for IL-2 activation. Substitution of phenylalanines for tyrosines at these positions abrogated Stat5 activation. This was observed for IL-2-induced activation when Jak3 was the only kinase transfected (Fig. 5F, lanes 4 and 8 versus lanes 2 and 6) and for the constitutive activation of Stat5A and Stat5B seen with Jak1 (Fig. 5G, lanes 2 and 4 versus lanes 1 and 3). As in Fig. 5A, endogenous Stat1 was activated by Jak1 (Fig. 5G), but not by Jak3 (Fig. 5F).


DISCUSSION

We have isolated cDNAs encoding two closely related human Stat5 proteins. Whereas only a single human Stat5 cDNA was previously identified, we have now shown that Stat5 DNA binding activity results from two closely related human proteins, Stat5A and Stat5B, analogous to the murine system. Both genes are located at chromosome 17q11.2, suggesting that they arose by tandem gene duplication. Interestingly, Stat5A and Stat5B are more similar to their murine homologues (34, 35) than to each other (Fig. 1).

Using antisera specific for Stat5A and Stat5B, we demonstrated that both Stat5A and Stat5B are activated by IL-2 in normal PBL, YT cells, and human T cell lymphotropic virus type I-transformed MT-2 cells and that both Stat5 proteins are involved in DNA binding. Stat5A and Stat5B migrate at different molecular masses, with Stat5B existing in at least two forms. These findings at least partially explain the heterogeneity observed previously in the mobility of Stat5(22, 23) .

Dimerization of IL-2Rbeta and (c) was previously shown to be essential for transducing IL-2-induced signals(28, 44) . We now demonstrate that in COS-7 cells, which constitutively express endogenous Jak1, we can reconstitute IL-2-induced Stat5 activation by transfecting Jak3 in addition to receptor chains, Stat5A, and/or Stat5B. Unexpectedly, high constitutive levels of Stat5 binding activity were observed with Jak1 (in both the presence and absence of Jak3); this implies that with supernormal levels of Jak1, there is direct activation of Stat5 by Jak1. Using this COS cell reconstitution system, we have established that activation of both Stat5A and Stat5B requires either Tyr-392 or Tyr-510 of IL-2Rbeta as a docking site. Moreover, we show that Tyr-694 of Stat5A and Tyr-699 of Stat5B are essential for the activation of these proteins.

Although the specific roles of Jak1 and Jak3 in IL-2 signaling remain unclear, in vitro experiments indicate that Jak1 can potently phosphorylate the IL-2Rbeta docking sites Tyr-392 and Tyr-510 (29) , whereas the importance of Jak3 is underscored by the lack of Stat6 activation in an Epstein-Barr virus-transformed B cell line derived from a Jak3-deficient patient with severe combined immunodeficiency (45) and in Jak3-deficient mice(46) . We therefore hypothesize that Jak3 may play a critical role in the phosphorylation of Stat5A and Stat5B, particularly since Stat5 and Jak3 can be coprecipitated in human T cell lymphotropic virus type I-transformed T cell lines, consistent with the possibility that they physically interact(47) .

Stat5 proteins can be activated by a variety of different cytokines or growth factors including prolactin, IL-2, IL-3, IL-7, IL-15, granulocyte-macrophage colony-stimulating factor, erythropoietin, growth hormone, and thrombopoietin. A number of these cytokines can reconstitute Stat5 binding activity in COS cells. Interestingly, however, although prolactin can activate transcription of a STAT-dependent reporter construct in these cells(36) , IL-2 cannot, even when both Stat3 and Stat5 are cotransfected (data not shown). Thus, cytokine-specific and/or in some cases tissue-specific signaling pathways may be involved in transcriptional activation by Stat5 proteins. Nonidentical sets of genes are induced by at least some of the cytokines that activate Stat5, indicating that Stat5 alone does not determine the specific biological effects of each cytokine.

It has recently been shown that serine phosphorylation of Stat3 is also required for the formation of a stable Stat3 dimer-DNA complex in a cell type- and binding site-dependent manner(48) , and both tyrosine phosphorylation and serine phosphorylation of Stat1 and Stat3 are essential for maximal interferon-induced transcription(49) , indicating important roles for both JAK kinases and serine kinases. In addition to activating a variety of tyrosine kinases(2) , IL-2 also activates other signaling molecules, including serine kinases (50) and Ras(51) . The COS-7 reconstitution system reported herein may be valuable in screening transfected gene products to help identify other signaling molecules required for mediating IL-2-dependent, Stat5-mediated transcription and in clarifying whether Stat5A homodimers, Stat5B homodimers, and Stat5A-Stat5B heterodimers regulate the same or different genes.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Lab. of Molecular Immunology, NHLBI, NIH, 9000 Rockville Pike, Bethesda, MD 20892-1674. Tel.: 301-496-0098; Fax: 301-402-0971.

(^1)
The abbreviations used are: IL-2, interleukin-2; IL-2R, interleukin-2 receptor; (c), common cytokine receptor -chain; PBL, peripheral blood lymphocyte(s); PHA, phytohemagglutinin; PCR, polymerase chain reaction; GAS, -interferon-activated site; kb, kilobases.


ACKNOWLEDGEMENTS

We thank J. Yodoi for YT and YT-1 cells; G. Poy for preparing synthetic peptides; J. N. Ihle for the murine Jak1 cDNA in pMLCMV; J. J. O'Shea for the human Jak3 cDNA; J. Bonafacino for pSX; A. Miyajima for pME18S and the murine Stat1, Stat2, Stat3, and Stat4 cDNAs in pME18S; M. Friedmann for preparing the IL-2Rbeta constructs containing mutated tyrosines; and S. Chen-Kiang, H. Young, and S. M. Russell for critical comments.


REFERENCES

  1. Takeshita, T., Asao, H., Ohtani, K., Ishii, N., Kumaki, S., Tanaka, N., Munakata, H., Nakamura, M., and Sugamura, K. (1992) Science 257, 379-382 [Medline] [Order article via Infotrieve]
  2. Taniguchi, T. (1995) Science 268, 251-255 [Medline] [Order article via Infotrieve]
  3. Leonard, W. J. (1994) Curr. Opin. Immunol. 6, 631-635 [CrossRef][Medline] [Order article via Infotrieve]
  4. Bazan, J. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6934-6938 [Abstract]
  5. Giri, J. G., Ahdieh, M., Eisenman, J., Shanebeck, K., Grabstein, K., Kumaki, S., Namen, A., Park, L. S., Cosman, D., and Anderson, D. (1994) EMBO J. 13, 2822-2830 [Abstract]
  6. Bamford, R. N., Grant, A. J., Burton, J. D., Peters, C., Kurys, G., Goldman, C. K., Brennan, J., Roessler, E., and Waldmann, T. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4940-4944 [Abstract]
  7. Kondo, M., Takeshita, T., Ishii, N., Nakamura, M., Watanabe, S., Arai, K., and Sugamura, K. (1993) Science 262, 1874-1877 [Medline] [Order article via Infotrieve]
  8. Russell, S. M., Keegan, A. D., Harada, N., Nakamura, Y., Noguchi, M., Leland, P., Friedmann, M. C., Miyajima, A., Puri, R. K., Paul, W. E., and Leonard, W. J. (1993) Science 262, 1880-1883 [Medline] [Order article via Infotrieve]
  9. Noguchi, M., Nakamura, Y., Russell, S. M., Ziegler, S. F., Tsang, M., Cao, X., and Leonard, W. J. (1993) Science 262, 1877-1880 [Medline] [Order article via Infotrieve]
  10. Kondo, M., Takeshita, T., Higuchi, M., Nakamura, M., Sudo, T., Nishikawa, S., and Sugamura, K. (1994) Science 263, 1453-1454 [Medline] [Order article via Infotrieve]
  11. Russell, S. M., Johnston, J. A., Noguchi, M., Kawamura, M., Bacon, C. M., Friedmann, M., Berg, M., McVicar, D. W., Witthuhn, B. A., Silvennoinen, O., Goldman, A. S., Schmalstieg, F. C., Ihle, J. N., O'Shea, J. J., and Leonard, W. J. (1994) Science 266, 1042-1045 [Medline] [Order article via Infotrieve]
  12. Kimura, Y., Takeshita, T., Kondo, M., Ishii, N., Nakamura, M., Van Snick, J., and Sugamura, K. (1995) Int. Immunol. 7, 115-120 [Abstract]
  13. Noguchi, M., Yi, H., Rosenblatt, H. M., Filipovich, A. H., Adelstein, S., Modi, W. S., McBride, O. W., and Leonard, W. J. (1993) Cell 73, 147-157 [Medline] [Order article via Infotrieve]
  14. Leonard, W. J., Noguchi, M., Russell, S. M., and McBride, O. W. (1994) Immunol. Rev. 138, 61-86 [Medline] [Order article via Infotrieve]
  15. Leonard, W. J. (1996) Annu. Rev. Med. 47, 229-239 [CrossRef][Medline] [Order article via Infotrieve]
  16. Johnston, J. A., Kawamura, M., Kirken, R. A., Chen, Y. Q., Blake, T. B., Shibuya, K., Ortaldo, J. R., McVicar, D. W., and O'Shea, J. J. (1994) Nature 370, 151-153 [CrossRef][Medline] [Order article via Infotrieve]
  17. Witthuhn, B. A., Silvennoinen, O., Miura, O., Lai, K. S., Cwik, C., Liu, E. T., and Ihle, J. N. (1994) Nature 370, 153-157 [CrossRef][Medline] [Order article via Infotrieve]
  18. Boussiotis, V. A., Barber, D. L., Nakarai, T., Freeman, G. J., Gribben, J. G., Bernstein, G. M., D'Andrea, A. D., Ritz, J., and Nadler, L. M. (1994) Science 266, 1039-1042 [Medline] [Order article via Infotrieve]
  19. Miyazaki, T., Kawahara, A., Fujii, H., Nakagawa, Y., Minami, Y., Liu, Z. J., Oishi, I., Silvennoinen, O., Witthuhn, B. A., Ihle, J. N., and Taniguchi, T. (1994) Science 266, 1045-1047 [Medline] [Order article via Infotrieve]
  20. Darnell, J. E., Jr., Kerr, I. M., and Stark, G. R. (1994) Science 264, 1415-1421 [Medline] [Order article via Infotrieve]
  21. Ihle, J. N. (1995) Nature 377, 591-594 [CrossRef][Medline] [Order article via Infotrieve]
  22. Lin, J.-X., Migone, T.-S., Tsang, M., Friedmann, M., Weatherbee, J. A., Zhou, L., Yamauchi, A., Bloom, E. T., Mietz, J., John, S., and Leonard, W. J. (1995) Immunity 2, 331-339 [Medline] [Order article via Infotrieve]
  23. Hou, J., Schindler, U., Henzel, W. J., Wong, S. C., and McKnight, S. L. (1995) Immunity 2, 321-329 [Medline] [Order article via Infotrieve]
  24. Yodoi, J., Teshigawara, K., Nikaido, T., Fukui, K., Noma, T., Honjo, T., Takigawa, M., Sasaki, M., Minato, N., Tsudo, M., Uchiyama, T., and Maeda, M. (1985) J. Immunol. 134, 1623-1630 [Abstract/Free Full Text]
  25. Wakao, H., Gouilleux, F., and Groner, B. (1994) EMBO J. 13, 2182-2191 [Abstract]
  26. Gnarra, J. R., Otani, H., Wang, M. G., McBride, O. W., Sharon, M., and Leonard, W. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3440-3444 [Abstract]
  27. Tory, K., Latif, F., Modi, W., Schmidt, L., Wei, M. H., Li, H., Cobler, P., Orcutt, M. L., Delisio, J., Geil, L., Zbar, B., and Lerman, M. I. (1992) Genomics 13, 275-286 [Medline] [Order article via Infotrieve]
  28. Nakamura, Y., Russell, S. M., Mess, S. A., Friedmann, M., Erdos, M., Francois, C., Jacques, Y., Adelstein, S., and Leonard, W. J. (1994) Nature 369, 330-333 [CrossRef][Medline] [Order article via Infotrieve]
  29. Friedmann, M. C., Migone, T.-S., Russell, S. M., and Leonard, W. J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2077-2082 [Abstract/Free Full Text]
  30. Lin, J.-X., Bhat, N. K., John, S., Queale, W. S., and Leonard, W. J. (1993) Mol. Cell. Biol. 13, 6201-6210 [Abstract]
  31. Fujii, H., Nakagawa, Y., Schindler, U., Kawahara, A., Mori, H., Gouilleux, F., Groner, B., Ihle, J. N., Minami, Y., Miyazaki, T., and Taniguchi, T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5482-5486 [Abstract]
  32. Gaffen, S. L., Lai, S. Y., Xu, W., Gouilleux, F., Groner, B., Goldsmith, M. A., and Greene, W. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7192-7196 [Abstract]
  33. Wakao, H., Harada, N., Kitamura, T., Mui, A. L.-F., and Miyajima, A. (1995) EMBO J. 14, 2527-2535 [Abstract]
  34. Mui, A. L.-F., Wakao, H., O'Farrell, A. M., Harada, N., and Miyajima, A. (1995) EMBO J. 14, 1166-1175 [Abstract]
  35. Azam, M., Erdjument-Bromage, H., Kreider, B. L., Xia, M., Quelle, F., Basu, R., Saris, C., Tempst, P., Ihle, J. N., and Schindler, C. (1995) EMBO J. 14, 1402-1411 [Abstract]
  36. Gouilleux, F., Pallard, C., Dusanter-Fourt, I., Wakao, H., Haldosen, L. A., Norstedt, G., Levy, D., and Groner, B. (1995) EMBO J. 14, 2005-2013 [Abstract]
  37. Pallard, C., Gouilleux, F., Benit, L., Cocault, L., Souyri, M., Levy, D., Groner, B., Gisselbrecht, S., and Dusanter-Fourt, I. (1995) EMBO J. 14, 2847-2856 [Abstract]
  38. Horvath, C. M., Wen, Z., and Darnell, J. E., Jr. (1995) Genes & Dev. 9, 984-994
  39. Schindler, U., Wu, P., Rothe, M., Brasseur, M., and McKnight, S. L. (1995) Immunity 2, 689-697 [Medline] [Order article via Infotrieve]
  40. Copeland, N. G., Gilbert, D. J., Schindler, C., Zhong, Z., Wen, Z., Darnell, J. E., Jr., Mui, A. L.-F., Miyajima, A., Quelle, F. W., Ihle, J. N., and Jenkins, N. A. (1995) Genomics 29, 225-228 [CrossRef][Medline] [Order article via Infotrieve]
  41. Yamamoto, K., Quelle, F. W., Thierfelder, W. E., Kreider, B. L., Gilbert, D. J., Jenkins, N. A., Copeland, N. G., Silvennoinen, O., and Ihle, J. N. (1994) Mol. Cell. Biol. 14, 4342-4349 [Abstract]
  42. Dusanter-Fourt, I., Muller, O., Ziemiecki, A., Mayeux, P., Drucker, B., Djiane, J., Wilks, A., Harpur, A. G., Fischer, S., and Gisselbrecht, S. (1994) EMBO J. 13, 2583-2591 [Abstract]
  43. Rui, H., Kirken, R. A., and Farrar, W. L. (1994) J. Biol. Chem. 269, 5364-5368 [Abstract/Free Full Text]
  44. Nelson, B. H., Lord, J. D., and Greenberg, P. D. (1994) Nature 369, 333-336 [CrossRef][Medline] [Order article via Infotrieve]
  45. Russell, S. M., Tayebi, N., Nakajima, H., Riedy, M. C., Roberts, J. L., Aman, M. J., Migone, T.-S., Noguchi, M., Markert, L. M., Buckley, R. H., O'Shea, J. J., and Leonard, W. J. (1995) Science 270, 797-800 [Abstract]
  46. Nosaka, T., van Deursen, J. M. A., Tripp, R. A., Thierfelder, W. E., Witthuhn, B. A., McMickle, A. P., Doherty, P. C., Grosveld, G. C., and Ihle, J. N. (1995) Science 270, 800-802 [Abstract]
  47. Migone, T.-S., Lin, J.-X., Cereseto, A., Mulloy, J. C., O'Shea, J. J., Franchini, G., and Leonard, W. J. (1995) Science 269, 79-81 [Medline] [Order article via Infotrieve]
  48. Zhang, X., Blenis, J., Li, H. C., Schindler, C., and Chen-Kiang, S. (1995) Science 267, 1990-1994 [Medline] [Order article via Infotrieve]
  49. Wen, Z., Zhong, Z., and Darnell, J. E., Jr. (1995) Cell 82, 241-250 [Medline] [Order article via Infotrieve]
  50. Turner, B., Rapp, U., App, H., Greene, M., Dobashi, K., and Reed, J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1227-1231 [Abstract]
  51. Izquierdo, M., and Cantrell, D. A. (1993) Eur. J. Immunol. 23, 131-135 [Medline] [Order article via Infotrieve]

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