Amino-terminal Signal Transducer and Activator of Transcription (STAT) Domains Regulate Nuclear Translocation and STAT Deactivation
1998; Elsevier BV; Volume: 273; Issue: 43 Linguagem: Inglês
10.1074/jbc.273.43.28049
ISSN1083-351X
AutoresInga Strehlow, Christian Schindler,
Tópico(s)Reproductive System and Pregnancy
ResumoThe first ∼100 amino acids of the STAT (signal transducer and activator oftranscription) family of transcription factors share a high degree of sequence similarity. To determine whether they encode a functionally conserved domain, amino-terminal chimeric STATs were created. These chimeric STATs share a number of properties with wild-type Stat1, including a predominately cytoplasmic pattern of expression in unstimulated cells. Upon stimulation with ligand, the chimeric STATs rapidly become tyrosine-phosphorylated, dimerize, and are able to bind DNA. They are also able to heterodimerize with coexpressed wild-type Stat1. Yet in contrast to wild-type Stat1, the chimeric STATs exhibit a marked defect in deactivation. Moreover, the persistence of active chimeras correlates directly with an inability to translocate to the nucleus. The defects both in nuclear translocation and in deactivation are rescued by heterodimerization with coexpressed wild-type Stat1. This study indicates that STAT amino termini provide a signal that is important for nuclear translocation and, subsequently, deactivation. It also suggests that deactivation may depend on a prior nuclear localization event. The first ∼100 amino acids of the STAT (signal transducer and activator oftranscription) family of transcription factors share a high degree of sequence similarity. To determine whether they encode a functionally conserved domain, amino-terminal chimeric STATs were created. These chimeric STATs share a number of properties with wild-type Stat1, including a predominately cytoplasmic pattern of expression in unstimulated cells. Upon stimulation with ligand, the chimeric STATs rapidly become tyrosine-phosphorylated, dimerize, and are able to bind DNA. They are also able to heterodimerize with coexpressed wild-type Stat1. Yet in contrast to wild-type Stat1, the chimeric STATs exhibit a marked defect in deactivation. Moreover, the persistence of active chimeras correlates directly with an inability to translocate to the nucleus. The defects both in nuclear translocation and in deactivation are rescued by heterodimerization with coexpressed wild-type Stat1. This study indicates that STAT amino termini provide a signal that is important for nuclear translocation and, subsequently, deactivation. It also suggests that deactivation may depend on a prior nuclear localization event. interferons Janus kinase interferon-γ activation sequence granulocyte colony-stimulating factor electrophoretic mobility shift assay whole cell extract phosphate-buffered saline signal transducer and activator of transcription. Cytokines mediate their pleiotropic effects on cells by binding to specific transmembrane-spanning receptors. These receptors transduce signals into the cell, culminating in the induction of new genes. Characterization of the ability of IFNs1 to rapidly induce new genes has led to the elucidation of the JAK-STAT (signaltransducer and activator oftranscription) signaling pathway, which is now known to transduce signals for other cytokines as well (1Schindler C. Darnell J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1636) Google Scholar, 2Strehlow I. Schindler C. Karupiah G. Gamma Interferon in Antiviral Defense. R. G. Landes Co., Austin, TX1997: 61-84Google Scholar). JAKs are receptor-associated tyrosine kinases that mediate ligand-dependent receptor phosphorylation. Receptor phosphotyrosyl residues are, in turn, specifically recognized by the SH2 domain of members of the STAT family of cytoplasmic transcription factors (3Stahl N. Farruggella T.J. Boulton T.G. Zhong Z. Darnell J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1352Crossref PubMed Scopus (862) Google Scholar, 4Yan H. Krishnan K. Greenlund A. Gupta S. Lim J.T. Schreiber R.D. Schindler C. Krolewski J.J. EMBO J. 1996; 15: 1064-1074Crossref PubMed Scopus (157) Google Scholar, 5Greenlund A.L. Farrar M.A. Viviano B.L. Schreiber R.D. EMBO J. 1994; 13: 1591-1600Crossref PubMed Scopus (371) Google Scholar). Once recruited to the receptor, STATs are activated by a single tyrosine phosphorylation event. Activated STATs are released from the receptor and then heterodimerize through the interaction between the phosphotyrosine of one STAT and the SH2 domain of another STAT (6Chen X. Winkemeier U. Zhao Y. Jeruzalmi D. Darnell J.E. Kuriyan J. Cell. 1998; 93: 827-839Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, 7Gupta S. Yan H. Wong L.H. Ralph S. Krolewski J. Schindler C. EMBO J. 1996; 15: 1075-1084Crossref PubMed Scopus (134) Google Scholar, 8Shuai K. Horvarth C.M. Tsai-Huang L.H. Quereshi S.A. Cowburn D. Darnell J.E. Cell. 1994; 76: 821-828Abstract Full Text PDF PubMed Scopus (677) Google Scholar, 9Becker S. Groner B. Müller C.W. Science. 1998; 394: 145-151Google Scholar). These active heterodimers translocate to the nucleus by a poorly understood mechanism and bind to a member of the GAS family of enhancers (1Schindler C. Darnell J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1636) Google Scholar, 10Schindler C. Shuai K. Prezioso V. Darnell J.E. Science. 1992; 257: 809-813Crossref PubMed Scopus (713) Google Scholar, 11Sekimoto T. Nakajima K. Tachibana T. Hirano T. Yoneda Y. J. Biol. Chem. 1996; 271: 31017-31020Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 12Sekimoto T. Imamoto N. Nakajima K. Tachibana T. Hirano T. Yoneda Y. EMBO J. 1997; 16: 7076-7077Crossref Scopus (306) Google Scholar). Seven STATs, ranging in size from ∼90 to ∼115 kDa, have been reported in mammals (1Schindler C. Darnell J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1636) Google Scholar, 2Strehlow I. Schindler C. Karupiah G. Gamma Interferon in Antiviral Defense. R. G. Landes Co., Austin, TX1997: 61-84Google Scholar, 13Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1258) Google Scholar). Sequence comparison of these STATs has led to the identification of several well conserved domains, the most highly conserved of which is the SH2 domain (7Gupta S. Yan H. Wong L.H. Ralph S. Krolewski J. Schindler C. EMBO J. 1996; 15: 1075-1084Crossref PubMed Scopus (134) Google Scholar, 8Shuai K. Horvarth C.M. Tsai-Huang L.H. Quereshi S.A. Cowburn D. Darnell J.E. Cell. 1994; 76: 821-828Abstract Full Text PDF PubMed Scopus (677) Google Scholar). Carboxyl-terminal to this domain is the tyrosine that becomes activated in response to ligand and subsequently mediates dimerization (10Schindler C. Shuai K. Prezioso V. Darnell J.E. Science. 1992; 257: 809-813Crossref PubMed Scopus (713) Google Scholar, 14Shuai K. Stark G.R. Kerr I.M. Darnell J.E. Science. 1993; 261: 1743-1744Crossref Scopus (678) Google Scholar, 15Improta T. Schindler C. Horvath C.M. Kerr I.M. Stark G.R. Darnell J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4776-4780Crossref PubMed Scopus (127) Google Scholar). Amino-terminal to this domain, but separated by an ∼80-amino acid linker region, is the DNA-binding domain (6Chen X. Winkemeier U. Zhao Y. Jeruzalmi D. Darnell J.E. Kuriyan J. Cell. 1998; 93: 827-839Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, 9Becker S. Groner B. Müller C.W. Science. 1998; 394: 145-151Google Scholar, 16Horvath C.M. Wen Z. Darnell J.E. Genes Dev. 1995; 9: 984-994Crossref PubMed Scopus (450) Google Scholar). There are several additional conserved amino-terminal domains whose structures suggest they may mediate interactions with other proteins. Of these, the first ∼100 amino acids are most conserved. Recent structural and functional studies suggest that this domain promotes cooperativity of DNA binding by mediating an interaction between two STAT dimers (6Chen X. Winkemeier U. Zhao Y. Jeruzalmi D. Darnell J.E. Kuriyan J. Cell. 1998; 93: 827-839Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, 9Becker S. Groner B. Müller C.W. Science. 1998; 394: 145-151Google Scholar, 17Vinkemeier U. Cohen S.L. Moarefi I. Chait B.T. Kuriyan J. Darnell J.E. EMBO J. 1996; 15: 5616-5626Crossref PubMed Scopus (247) Google Scholar, 18Vinkemeier U. Moarefi I. Darnell J.E. Kuriyan J. Science. 1998; 279: 1048-1052Crossref PubMed Scopus (210) Google Scholar). As expected, mutagenesis of an invariant tryptophan (Trp-37 for Stat1) that is critical for this interaction abrogates cooperativity in DNA binding (18Vinkemeier U. Moarefi I. Darnell J.E. Kuriyan J. Science. 1998; 279: 1048-1052Crossref PubMed Scopus (210) Google Scholar). Mutation of another highly conserved residue in this interaction domain (Arg-31) has been shown to correlate with persistent tyrosine phosphorylation (19Shuai K. Liao J. Song M. Mol. Cell. Biol. 1996; 16: 4932-4941Crossref PubMed Scopus (131) Google Scholar). Although the structure of this region does not support a role for Arg-31 in mediating an interaction with a phosphatase, as initially argued, these observations do suggest that the amino-terminal domain is likely to serve an important regulatory function(s) in addition to cooperativity of DNA binding. To determine whether these highly conserved amino-terminal domains are functionally conserved and to evaluate the possibility that they may participate in other aspects of STAT signaling, chimeric STATs were generated. As anticipated, when the amino terminus of Stat1 (amino acids 1–129) was replaced with the homologous regions of Stat2 or Stat5, many functional properties were conserved. However, these chimeras exhibited a marked defect in nuclear translocation and deactivation. Chimeric receptors were generated by introducing DNA encoding amino acids 437–518 from the endodomain and transmembrane domain of the human IFN-α receptor α-chain (20Yan H. Krishnan K. Lim J.T. Contillo L.G. Krolewski J.J. Mol. Cell. Biol. 1996; 16: 2082-2974Google Scholar) into RcCMV (Invitrogen) encoding the 2200-base pair ectodomain of the human G-CSF receptor (21Ziegler S.P. Bird T.A. Morella K.K. Mosley B. Gearing D.P. Baumann H. Mol. Cell. Biol. 1993; 13: 2384-2390Crossref PubMed Scopus (111) Google Scholar). Oligonucleotides encoding the Stat1-binding/recruitment site (TSFGYDKPHV) from the human interferon-γ receptor α-chain (5Greenlund A.L. Farrar M.A. Viviano B.L. Schreiber R.D. EMBO J. 1994; 13: 1591-1600Crossref PubMed Scopus (371) Google Scholar) and a triple Myc tag (3Stahl N. Farruggella T.J. Boulton T.G. Zhong Z. Darnell J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1352Crossref PubMed Scopus (862) Google Scholar) were added to the 3′-end of the receptor chimera to yield the mature construct G/aRα. The CH2/1 construct was generated by introducing a polymerase chain reaction product encoding amino acids 1–140 of Stat2, through blunt end cloning, into an RcCMV-based expression construct encoding amino acids 130–750 of Stat1. Similarly, the STAT CH5/1 construct was prepared by the introduction of a polymerase chain reaction product encoding amino acids 1–129 of Stat5b into the same Stat1 expression construct (i.e. amino acids 130–750). Each of these constructs was confirmed by sequencing. The generation of ΔNStat1 (amino acids 135–750) has been previously described (22Meraz M.A. White J.M. Sheehan K.C. Bach E.A. Rodig S.J. Dighe A.S. Kaplan D.H. Riley J.K. Greenlund A.C. Campbell D. Carver-Moore K. DuBois R.N. Clark R. Aguet M. Schreiber R.D. Cell. 1996; 84: 431-442Abstract Full Text Full Text PDF PubMed Scopus (1377) Google Scholar). The human kidney fibroblast cell line 293 was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Life Technologies, Inc.). For transfection, 293 cells were pretreated for 5 min with 25 mm chloroquine (Sigma) and then transfected with 10 μg of the STAT constructs and 10 μg of G/aRα by the calcium phosphate method (23Pear E.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2275) Google Scholar). After 24 h, transfected cells were reseeded and, after another 24 h, stimulated with human recombinant G-CSF (250 ng/ml; Amgen) or IFN-α (1000 units/ml; Genentech). As indicated, in some studies, staurosporine (dissolved in Me2SO; Sigma) was added to a final concentration of 500 nm. Electrophoretic mobility shift assays (EMSAs) were carried out with an IRF1 GAS probe (gatcGATTTCCCCGAAAT; Oligos Etc.) as described previously (24Bonni A. Frank D.A. Schindler C. Greenberg M.E. Science. 1993; 262: 1575-1579Crossref PubMed Scopus (162) Google Scholar, 25Pine R. Canova A. Schindler C. EMBO J. 1994; 13: 158-167Crossref PubMed Scopus (339) Google Scholar, 26Rothman P. Kreider B. Azam M. Levy D. Wegenka U. Eilers A. Decker T. Horn F. Kashleva H. Ihle J. Schindler C. Immunity. 1994; 1: 457-468Abstract Full Text PDF PubMed Scopus (76) Google Scholar). Cytoplasmic, nuclear, or whole cell extracts (WCEs) were made as reported previously (10Schindler C. Shuai K. Prezioso V. Darnell J.E. Science. 1992; 257: 809-813Crossref PubMed Scopus (713) Google Scholar, 25Pine R. Canova A. Schindler C. EMBO J. 1994; 13: 158-167Crossref PubMed Scopus (339) Google Scholar, 27Eilers A. Seegert D. Schindler C. Baccarini M. Decker T. Mol. Cell. Biol. 1993; 13: 3245-3254Crossref PubMed Scopus (47) Google Scholar). For supershifts, 0.6 μl of anti-STAT antibodies were added to a standard DNA binding reaction (12.5 μl for 20 min at 25 °C) and incubated at 4 °C for an additional hour prior to EMSA. In some studies, cytoplasmic extracts were treated with calf intestinal phosphatase (Buffer 2, New England Biolabs Inc.) for 1 h at 37 °C prior to evaluation by EMSA. Immunoprecipitations and immunoblotting were performed as described previously (28Azam M. Erdjument-Bromage H. Kreider B.L. Xia M. Quelle F. Basu R. Saris C. Tempst P. Ihle J.N. Schindler C. EMBO J. 1995; 14: 1402-1411Crossref PubMed Scopus (298) Google Scholar). Briefly, immunoprecipitates were fractionated by 7% SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and immunoblotted with the following: a phosphotyrosine-specific antibody (4G10; Upstate Biotechnology, Inc.), a Myc epitope-specific antibody (9E10; Santa Cruz Biotechnology), a Tyk2-specific antibody (Santa Cruz Biotechnology), or STAT-specific antibodies. STAT-specific antibodies included those recognizing the amino terminus of Stat1 (N1 (29Schindler C. Fu X.-Y. Improta T. Aebersold R. Darnell J.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7836-7839Crossref PubMed Scopus (539) Google Scholar)), the carboxyl terminus of Stat1 (C1 (29Schindler C. Fu X.-Y. Improta T. Aebersold R. Darnell J.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7836-7839Crossref PubMed Scopus (539) Google Scholar)), the amino terminus of Stat2 (monoclonal N2; Transduction Laboratories); and the amino terminus of Stat5 (N5, prepared against a glutathione S-transferase fusion protein of Stat5b amino acids 1–49 (28Azam M. Erdjument-Bromage H. Kreider B.L. Xia M. Quelle F. Basu R. Saris C. Tempst P. Ihle J.N. Schindler C. EMBO J. 1995; 14: 1402-1411Crossref PubMed Scopus (298) Google Scholar)). N5 represents an IgG fraction prepared from crude serum (Pierce). Immunoblotted bands were detected by ECL as specified by the manufacturer (Amersham Pharmacia Biotech). For sequential Western blots, filters were stripped in 2% SDS, 65 mm Tris (pH 6.7), and 100 mmβ-mercaptoethanol for 45 min at 50 °C. Cells were rinsed twice in phosphate-buffered saline (PBS) and fixed in 2% formaldehyde/PBS. After two additional PBS rinses, cells were treated with 1% Triton X-100 (in PBS; Sigma) for 2 min and then washed in 0.1% Tween 20 (in PBS; Sigma). Nonspecific protein adsorption was blocked by a 30-min incubation with 2% bovine serum albumin/PBS. Samples were incubated with primary antibody (C1; 1:200 in 2% bovine serum albumin/PBS) for 1 h (22 °C), washed, and then incubated with secondary antibody (Cy3-conjugated donkey anti-rabbit antibody, 1:300; Jackson ImmunoResearch Laboratories, Inc.) for 30 min. These samples were washed and examined at a magnification of ×400 under a Zeiss LSM410 laser scanning confocal microscope. 293 cells were selected to evaluate the function of mutant STATs because of their high transfection efficiency. However, as recent studies suggested that the amino-terminal domains of STATs may be required for a productive interaction with an appropriate receptor (30Li X. Leung S. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1997; 17: 2048-2956Crossref PubMed Scopus (162) Google Scholar), we set out to design a generic receptor that would activate wild-type and amino-terminal chimeric STATs with equivalent efficiency. The extracellular domain of the G-CSF receptor was selected as the ligand-binding domain because only one type of receptor subunit is required for ligand binding and because of its limited pattern of tissue expression (e.g. not in 293 cells (21Ziegler S.P. Bird T.A. Morella K.K. Mosley B. Gearing D.P. Baumann H. Mol. Cell. Biol. 1993; 13: 2384-2390Crossref PubMed Scopus (111) Google Scholar)). The endodomain included the minimal region of the IFN-α receptor α-chain (IFNAR1) implicated in JAK (i.e. Tyk2) binding/activation (20Yan H. Krishnan K. Lim J.T. Contillo L.G. Krolewski J.J. Mol. Cell. Biol. 1996; 16: 2082-2974Google Scholar, 31Colamonici O.R. Uyttendaele H. Domanski P. Yan H. Krolewski J.J. J. Biol. Chem. 1994; 269: 3518-3522Abstract Full Text PDF PubMed Google Scholar) and the 10-amino acid Stat1 recruitment/activation motif from the IFN-γ receptor α-chain (5Greenlund A.L. Farrar M.A. Viviano B.L. Schreiber R.D. EMBO J. 1994; 13: 1591-1600Crossref PubMed Scopus (371) Google Scholar). This chimeric receptor is referred to as G/aRα. To test the G/aRα receptor functionally, it was introduced into 293 cells by transient transfection and then stimulated with G-CSF. When the chimeric receptor was collected from transfected cells by virtue of its carboxyl-terminal Myc epitope tag (3Stahl N. Farruggella T.J. Boulton T.G. Zhong Z. Darnell J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1352Crossref PubMed Scopus (862) Google Scholar), it was found to be tyrosine-phosphorylated in response to ligand (Fig. 1 B). Moreover, activated Tyk2 co-immunoprecipitated with the receptor. Activation of Tyk2 was also evaluated directly. Consistent with previous studies (20Yan H. Krishnan K. Lim J.T. Contillo L.G. Krolewski J.J. Mol. Cell. Biol. 1996; 16: 2082-2974Google Scholar, 32Barbieri G.L. Velazquez L. Scrobogna M. Fellous M. Pellegrini S. Eur. J. Biochem. 1994; 223: 427-435Crossref PubMed Scopus (56) Google Scholar), a basal level of phosphorylated Tyk2 was observed in unstimulated cells, which then increased upon stimulation with G-CSF (Fig. 1 B). Stimulation of transfected cells with IFN-α, which activates the endogenous type I receptor, yielded a similar level of Tyk2 phosphorylation. These results demonstrate that the chimeric receptor and associated Tyk2 are activated in response to G-CSF in 293 cells. As anticipated, this receptor promotes the G-CSF-dependent activation of either endogenous or cotransfected Stat1 (see below). Experiments with control receptors demonstrated that the Stat1 recruitment tyrosine motif is required for STAT activation (data not shown). These results indicate that this chimeric receptor will be a valuable tool for the activation of transfected Stat1 chimeras. To test whether the conserved ∼100 amino acids at the amino terminus of STATs mediate a general or perhaps a more STAT-specific function(s), chimeric STAT molecules were prepared. To generate these chimeras, the amino terminus of Stat1 was carefully replaced by the homologous regions of Stat2 or Stat5 (Fig. 2 A). Amino acid 130 was selected for the site of fusion because it is poorly conserved and appears to form a random coil (6Chen X. Winkemeier U. Zhao Y. Jeruzalmi D. Darnell J.E. Kuriyan J. Cell. 1998; 93: 827-839Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, 9Becker S. Groner B. Müller C.W. Science. 1998; 394: 145-151Google Scholar, 17Vinkemeier U. Cohen S.L. Moarefi I. Chait B.T. Kuriyan J. Darnell J.E. EMBO J. 1996; 15: 5616-5626Crossref PubMed Scopus (247) Google Scholar, 18Vinkemeier U. Moarefi I. Darnell J.E. Kuriyan J. Science. 1998; 279: 1048-1052Crossref PubMed Scopus (210) Google Scholar). We predicted that if the function(s) encoded by this domain were conserved, then the chimeric molecules would behave similarly to wild-type Stat1. But, if they encoded STAT-specific functions, then these molecules should be functionally crippled. First, we determined whether these chimeric STATs were activated by the G/aRα receptor. Extracts were prepared from 293 cells cotransfected with both constructs, either before or after stimulation with G-CSF, and then evaluated by EMSA (Fig. 2 B). 293 cells transfected with G/aRα alone exhibited activation of endogenous Stat1α. The level of Stat1 DNA-binding activity increased when Stat1 was cotransfected. Surprisingly, cells cotransfected with the 2/1 or 5/1 chimeras (i.e. CH2/1 and CH5/1) demonstrated a more robust DNA-binding activity than wild-type Stat1 (either endogenous or recombinant). Moreover, the chimeric STATs exhibited a distinct mobility. Immunoblotting these extracts with a Stat1 carboxyl terminus-specific antibody (C1; an epitope that is present in each STAT construct) confirmed that each of these proteins was overexpressed to a similar level (Fig. 2 C). To further characterize these DNA-binding complexes, they were probed with antibodies directed either against the conserved Stat1 carboxyl terminus (C1) or their unique amino termini (N1 for Stat1, N2 for Stat2, and N5 for Stat5 (Fig. 2 D). Each of the chimeric complexes was recognized (i.e. supershifted or blocked) by C1. As anticipated, only the 5/1 chimera (CH5/1) was recognized by N5. Similarly, only the 2/1 chimera (CH2/1) interacted, albeit modestly, with N2. (N2 is a monoclonal antibody and not very effective at supershifting.) These studies demonstrate that chimeric STATs, activated through stimulation of a cotransfected receptor, each give rise to a novel DNA-binding complex. Moreover, although these STATs are all expressed at equivalent levels, the chimeric STATs give rise to more intense shift bands. The initial characterization of chimeric STAT DNA-binding activity was done 30 min after stimulation. To determine whether the observed differences in DNA-binding activity were dependent on the duration of stimulation, a kinetic study was undertaken. Transfected cells were stimulated with G-CSF for 5, 30, and 180 min. Consistent with published studies (33Haspel R.L. Salditt-Georgieff M. Darnell J.E. EMBO J. 1996; 15: 6262-6268Crossref PubMed Scopus (272) Google Scholar, 34Shuai K. Schindler C. Prezioso V. Darnell J.E. Science. 1992; 258: 1808-1812Crossref PubMed Scopus (652) Google Scholar), wild-type Stat1 was maximally phosphorylated by 5 min. Densitometric evaluation indicated that there was a 2.5–3-fold loss of DNA-binding activity by 180 min (Fig. 3 A). Evaluation of the chimeric STATs revealed some notable differences. Early after stimulation, chimeric STATs were phosphorylated to similar level as Stat1. However, their level of phosphorylation continued to increase at 30 min and exhibited little decay at 180 min after stimulation. Analogous results were obtained when phosphorylation was evaluated by a less sensitive immunoblotting assay (Fig. 3 B). As anticipated, the immunoblotting assay found a decrease in the level of Stat1 phosphorylation after 180 min of stimulation. In contrast, the chimeric STATs remained heavily phosphorylated at this time point. However, this assay failed to demonstrate an increase in chimeric STAT phosphorylation at 180 min. Rather, there was a modest decrease. This suggests that changes other than those in the level of tyrosine phosphorylation (e.g. structural; see below) may contribute to the increase in DNA-binding activity. A faster migrating band (Fig. 3 B, left panel, labeled NS) represents a very low abundance, amino-terminally truncated isoform of Stat1 that can be detected only after substantial enrichment by immunoprecipitation from 293 cell extracts (data not shown). The filter was then reprobed with STAT-specific antibodies C1, N2, and N5 to demonstrate that the transfected STATs were immunoprecipitated to the same extent. These results demonstrate equivalent levels of phosphorylation at early times and suggest that wild-type and chimeric STATs are equipotent substrates for the JAK kinases. The relative increase in the amount of activated (i.e. phosphorylated) chimeric STAT is consistent with a process of accumulation (i.e. a defect in deactivation; see below). To determine whether the apparent defect in deactivation was due to either the presence of new amino-terminal sequences (i.e.Stat2 or Stat5) or the loss of Stat1 amino-terminal sequences, an amino-terminal Stat1 deletion mutant (i.e. ΔNStat1 (22Meraz M.A. White J.M. Sheehan K.C. Bach E.A. Rodig S.J. Dighe A.S. Kaplan D.H. Riley J.K. Greenlund A.C. Campbell D. Carver-Moore K. DuBois R.N. Clark R. Aguet M. Schreiber R.D. Cell. 1996; 84: 431-442Abstract Full Text Full Text PDF PubMed Scopus (1377) Google Scholar)) was examined. Evaluation of the kinetics of G-CSF-stimulated ΔNStat1 demonstrated a prolonged pattern of activation (i.e. DNA binding), analogous to what was observed for the chimeric STATs (Fig. 3 C). However, the level of ΔNStat1 activation was even higher and more rapid than observed for the chimeric STATs. Immunoblotting studies confirmed that ΔNStat1 was expressed at levels similar to wild-type and chimeric Stat1 (data not shown). These observations indicate that a loss of Stat1 amino-terminal sequences leads to an increase in the level of phosphorylation. The domain(s) encoded in the amino terminus of Stat2 or Stat5 do not compensate for this loss. Recent studies demonstrating that the duration of ligand-stimulated Stat1 DNA-binding activity is sensitive to kinase inhibitors indicated that prolonged DNA binding is dependent on continuous JAK activity (33Haspel R.L. Salditt-Georgieff M. Darnell J.E. EMBO J. 1996; 15: 6262-6268Crossref PubMed Scopus (272) Google Scholar). Since chimeric STATs bind DNA long after JAK activity is likely to have decayed, their prolonged biological activity appears to be independent of kinase activity. To test this, the kinetics of wild-type and chimeric Stat1 DNA-binding activities were evaluated in the presence and absence of staurosporine, a JAK inhibitor (Fig. 4 A). Consistent with published studies, the DNA-binding activity of wild-type Stat1 was lost after 1 h of staurosporine treatment (Fig. 4 A, lane 5). In contrast, the DNA-binding activity of the chimeric STATs was only modestly affected by the addition of staurosporine (Fig. 4 A, lanes 10 and 15), indicating that only a fraction of the prolonged DNA-binding activity can be attributed to persistent kinase activity. Rather, these observations imply that an important cause for the increased amount of phosphorylated chimeric STATs is also due to a defect in deactivation. The preceding studies indicated that chimeric STATs accumulate in a phosphorylated form due to a defect in deactivation. However, they failed to provide any insight into whether this can be attributed to a structural change and/or a failure to localize to an appropriate compartment (e.g. where phosphatases may be active). Recently published studies suggested that a conserved amino-terminal STAT domain may promote interactions with a phosphatase (19Shuai K. Liao J. Song M. Mol. Cell. Biol. 1996; 16: 4932-4941Crossref PubMed Scopus (131) Google Scholar), but the crystal structure does not support this notion (18Vinkemeier U. Moarefi I. Darnell J.E. Kuriyan J. Science. 1998; 279: 1048-1052Crossref PubMed Scopus (210) Google Scholar). Moreover, a swap between two conserved domains would not be expected to affect a general function like the ability of STATs to interact with a phosphatase. Evaluation of the mobility of the chimeric STAT·DNA complexes indicated they differed from that of wild-type Stat1 (Figs. Figure 2, Figure 3, Figure 4). This suggested that the chimeras have undergone a structural change, at least when bound to DNA. Yet, most STAT functions were unaffected (e.g. the ability to productively interact with the receptor-kinase complex, to dimerize, or to bind DNA), indicating that the structural change was subtle. To crudely probe the structural properties of wild-type and chimeric Stat1, they were treated with three doses of a potent nonspecific phosphatase (Fig. 4 B). The DNA-binding activity of wild-type Stat1 was completely abrogated by 1 unit of phosphatase. In contrast, the 2/1 chimera had only a limited susceptibility to this phosphatase. A residual amount of DNA-binding activity was evident even after exposure to high doses of phosphatase. As anticipated, phosphatase activity was sensitive to sodium orthovanadate. Although these observations do not address the ability of chimeric STATs to potentially interact with native phosphatases, they do suggest that chimeric STATs exhibit a structural change that affects their ability to be nonspecifically dephosphorylated in vitro. Next, we examined the possibility that the accumulation of phosphorylated chimeric STATs might be secondary to failure to localize to an appropriate cellular compartment. Since previous studies had suggested that STATs may be dephosphorylated in the nucleus (35David M. Grimely P.M. Finbloom D.S. Larner A.C. Mol. Cell. Biol. 1993; 13: 7515-7521Crossref PubMed Scopus (102) Google Scholar), we considered a defect in nuclear translocation first. A sensitive immunofluorescence assay was selected to evaluate the ability of wild-type and chimeric STATs to translocate to the nucleus (10Schindler C. Shuai K. Prezioso V. Darnell J.E. Science. 1992; 257: 809-813Crossref PubMed Scopus (713) Google Scholar). In an effort to have the nuclear translocation of endogenous Stat1 (i.e. in nontransfected
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