A Single Residue Modulates Tyrosine Dephosphorylation, Oligomerization, and Nuclear Accumulation of Stat Transcription Factors

Abstract

The NH2 terminus of Stat proteins forms a versatile protein interaction domain that is believed to use discrete surfaces to mediate oligomerization and tyrosine dephosphorylation of Stat dimers. Here we show for Stat1 and Stat5a/b that these interfaces overlap and need to be reassigned to an unrelated region of the N-domain. Unexpectedly, our study showed for Stat1 that defective oligomerization of DNA-bound dimers was associated with prolonged interferon-induced nuclear accumulation. This uncoupling of DNA binding and nuclear retention was explained by the concomitant dephosphorylation deficiency that both Stat1 and Stat5a/b have in common and that for Stat1 was due to defective dephosphorylation by the phosphatase TC45. Furthermore, diminished N-domain-mediated oligomerization affected transcriptional activation by both Stat1 and Stat5a/b in a promoter-specific manner. DNA binding analysis indicated that oligomerization of Stats on DNA may be common, irrespective of the presence of multiple canonical binding sites. Accordingly, also transcription from promoters with only a single discernable γ-activated sequence site was negatively effected by reduced tetramerization. Thus, these results indicate that defective oligomerization cannot generally be compensated for by enhanced tyrosine phosphorylation and prolonged nuclear accumulation. In addition, these data clarify the role of DNA binding in nuclear retention of Stat1. The NH2 terminus of Stat proteins forms a versatile protein interaction domain that is believed to use discrete surfaces to mediate oligomerization and tyrosine dephosphorylation of Stat dimers. Here we show for Stat1 and Stat5a/b that these interfaces overlap and need to be reassigned to an unrelated region of the N-domain. Unexpectedly, our study showed for Stat1 that defective oligomerization of DNA-bound dimers was associated with prolonged interferon-induced nuclear accumulation. This uncoupling of DNA binding and nuclear retention was explained by the concomitant dephosphorylation deficiency that both Stat1 and Stat5a/b have in common and that for Stat1 was due to defective dephosphorylation by the phosphatase TC45. Furthermore, diminished N-domain-mediated oligomerization affected transcriptional activation by both Stat1 and Stat5a/b in a promoter-specific manner. DNA binding analysis indicated that oligomerization of Stats on DNA may be common, irrespective of the presence of multiple canonical binding sites. Accordingly, also transcription from promoters with only a single discernable γ-activated sequence site was negatively effected by reduced tetramerization. Thus, these results indicate that defective oligomerization cannot generally be compensated for by enhanced tyrosine phosphorylation and prolonged nuclear accumulation. In addition, these data clarify the role of DNA binding in nuclear retention of Stat1. The signal transducer and activator of transcription (Stat) 1The abbreviations used are: Stat, signal transducer and activator of transcription; Jak, Janus kinase; SH2, Src homology domain 2; GAS, γ-activated sequence; FRAP, fluorescence recovery after photobleaching; IFN, interferon; WT, wild type; RT, reverse transcription; IL, interleukin; GFP, green fluorescent protein; PRRIII, positive regulatory region III. proteins comprise an evolutionary conserved family of transcription factors (1Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11767-11769Google Scholar). Canonical signaling through the Janus tyrosine kinase (Jak)/Stat pathway begins at the cell membrane with the engagement of cytokines with their cognate receptor (2Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 69-74Google Scholar). This triggers the autophosphorylation on tyrosines of non-covalently attached Jak kinases, which also phosphorylate signature tyrosine residues in the intracellular receptor tails thus providing phosphotyrosine docking sites for the Stat SH2 domain (3Leaman D.W. Leung S. Li X. Stark G.R. FASEB J. 1996; 10: 1578-1588Google Scholar). The bound Stat monomers detach from their receptor docking sites after phosphorylation of a single tyrosine residue at their COOH terminus and form high avidity reciprocal homo- or heterodimers (4Shuai K. Stark G.R. Kerr I.M. Darnell Jr., J.E. Science. 1993; 261: 1744-1776Google Scholar, 5Shuai K. Horvath C.M. Huang L.H. Qureshi S.A. Cowburn D. Darnell Jr., J.E. Cell. 1994; 76: 821-828Google Scholar, 6Greenlund A.C. Morales M.O. Viviano B.L. Yan H. Krolewski J. Schreiber R.D. Immunity. 1995; 2: 677-687Google Scholar). This sequence of events is commonly referred to as “Stat activation” and within minutes triggers the accumulation of Stat dimers in the nucleus due to their inability to leave this compartment (7Meyer T. Marg A. Lemke P. Wiesner B. Vinkemeier U. Genes Dev. 2003; 17: 1992-2005Google Scholar). Here they can bind to palindromic DNA recognition sites (GAS) and directly induce transcription (8Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Google Scholar). The products of seven different Stat genes and various splice variants thereof can be found in mammalian cells. Gene inactivation experiments using homologous recombination have revealed that the most striking phenotypes are associated with signal transduction by particular cytokines. Stat1 is required for interferon-α (IFNα) and IFNγ signaling, and mice and humans lacking Stat1 are extremely sensitive to viral and microbial infection (9Dupuis S. Jouanguy E. Al-Hajjar S. Fieschi C. Al-Mohsen I.Z. Al-Jumaah S. Yang K. Chapgier A. Eidenschenk C. Eid P. Al Ghonaium A. Tufenkeji H. Frayha H. Al-Gazlan S. Al-Rayes H. Schreiber R.D. Gresser I. Casanova J.L. Nat. Genet. 2003; 33: 388-391Google Scholar, 10Durbin J.E. Hackenmiller R. Simon M.C. Levy D.E. Cell. 1996; 84: 443-450Google Scholar, 11Meraz 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: 431-442Google Scholar). The two highly related Stat5a and Stat5b proteins (∼96% sequence identity) are activated by a wide range of different cytokines such as interleukin 2 (IL-2), IL-7, prolactin, and growth factors. Accordingly, gene ablation experiments uncovered mammary gland defects, growth abnormalities, and an absent IL-2 response (12Liu X. Robinson G.W. Wagner K.U. Garrett L. Wynshaw-Boris A. Hennighausen L. Genes Dev. 1997; 15: 179-186Google Scholar, 13Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Google Scholar, 14Udy G.B. Towers R.P. Snell R.G. Wilkins R.J. Park S.H. Ram P.A. Waxman D.J. Davey H.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7239-7244Google Scholar). Stat proteins consist of a large core domain including the DNA binding module (15Horvath C.M. Wen Z. Darnell Jr., J.E. Genes Dev. 1995; 15: 984-994Google Scholar) and the SH2 domain responsible for dimer formation (5Shuai K. Horvath C.M. Huang L.H. Qureshi S.A. Cowburn D. Darnell Jr., J.E. Cell. 1994; 76: 821-828Google Scholar). The core is connected through a proteolytically cleavable peptide to the smaller N-domain, which engages in a multitude of protein-protein interactions. The N-domain mediates cooperative DNA binding of Stat dimers (16Li X. Leung S. Burns C. Stark G.R. Biochimie (Paris). 1998; 80: 703-710Google Scholar, 17John S. Vinkemeier U. Soldaini E. Darnell Jr., J.E. Leonard W.J. Mol. Cell. Biol. 1999; 19: 1910-1918Google Scholar, 18Vinkemeier U. Cohen S.L. Moarefi I. Chait B.T. Kuriyan J. Darnell Jr., J.E. EMBO J. 1996; 15: 5616-5626Google Scholar, 19Xu X. Sun Y.L. T. Science. 1996; Scholar, X. Darnell Jr., J.E. J. Biol. Scholar) and their tyrosine dephosphorylation K. J. Mol. Cell. Biol. 1996; Scholar, I. C. J. Biol. 1998; Scholar). The of the N-domain an U. Moarefi I. Darnell Jr., J.E. Kuriyan J. Science. 1998; Scholar). In the N-domain forms two binding surfaces are in the of the indicated that the of of these surfaces as responsible for cooperative DNA binding is X. R. Vinkemeier U. van Darnell Jr., J.E. Kuriyan J. Sci. 2003; Scholar). unrelated region of the N-domain by to be for the dephosphorylation of as of an residue K. J. Mol. Cell. Biol. 1996; Scholar). this the that the that this domain and thus may the of this at the interfaces that cooperative DNA binding and tyrosine dephosphorylation of the Stats we these and for Stat1 and Stat5a/b that the two interfaces data that DNA is for nuclear retention of Stat proteins and a promoter-specific need for cooperative DNA binding in gene and and as A. T. van M. Vinkemeier U. Proc. Natl. Acad. Sci. U. S. A. Scholar). of was with 2 IL-2 Stat1 proteins was with as (7Meyer T. Marg A. Lemke P. Wiesner B. Vinkemeier U. Genes Dev. 2003; 17: 1992-2005Google Scholar, T. A. I. van M. Vinkemeier U. EMBO J. Scholar). and and have (7Meyer T. Marg A. Lemke P. Wiesner B. Vinkemeier U. Genes Dev. 2003; 17: 1992-2005Google Scholar, 17John S. Vinkemeier U. Soldaini E. Darnell Jr., J.E. Leonard W.J. Mol. Cell. 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T. van M. Vinkemeier U. Proc. Natl. Acad. Sci. U. S. A. and the at are the are sites are the was for at a of the the was at for as In of a was and with the for at and dephosphorylation was as after with IFNγ or IL-2 for and the was with or or as or at the indicated after of and with with and cell by the cell in of for of was on and as A. T. van M. Vinkemeier U. Proc. Natl. Acad. Sci. U. S. A. Scholar) using a or a from or a or Stat1 In dephosphorylation for with Stat1 dimers and of and the indicated of Stat1 as (7Meyer T. Marg A. Lemke P. Wiesner B. Vinkemeier U. Genes Dev. 2003; 17: 1992-2005Google Scholar). Stat1 was at a of or The was by after in and of and Stat5a proteins in and as (7Meyer T. Marg A. Lemke P. Wiesner B. Vinkemeier U. Genes Dev. 2003; 17: 1992-2005Google Scholar, E. S. S. J. U. Leonard W.J. Mol. Biol. Scholar). Stat5a was by an kinase in the E. S. S. J. U. Leonard W.J. Mol. Biol. Scholar). In tyrosine phosphorylation with growth receptor and of Stat1 as (7Meyer T. Marg A. Lemke P. Wiesner B. Vinkemeier U. Genes Dev. 2003; 17: 1992-2005Google Scholar). of the was from and the of the wild Stat5a which was the was by at the residue and by DNA sequence Stat5a and to be by analysis using was in and as (7Meyer T. Marg A. Lemke P. Wiesner B. Vinkemeier U. Genes Dev. 2003; 17: 1992-2005Google Scholar). and after and as (7Meyer T. Marg A. Lemke P. Wiesner B. Vinkemeier U. Genes Dev. 2003; 17: 1992-2005Google Scholar). analysis was as T. A. I. van M. Vinkemeier U. EMBO J. Scholar) with Stat1 or and the and and and and The N-domain DNA of Stat1 and of Stat oligomerization from our and have indicated that a single residue of that is in the of N-domain dimers of Stats S. Vinkemeier U. Soldaini E. Darnell Jr., J.E. Leonard W.J. Mol. Cell. Biol. 1999; 19: 1910-1918Google Scholar, X. Darnell Jr., J.E. J. Biol. Scholar, U. Moarefi I. Darnell Jr., J.E. Kuriyan J. Science. 1998; Scholar). in with the N-domain that of and of the N-domain X. R. Vinkemeier U. van Darnell Jr., J.E. Kuriyan J. Sci. 2003; and the found to be with their in a or in IL-2 S. Vinkemeier U. Soldaini E. Darnell Jr., J.E. Leonard W.J. Mol. Cell. Biol. 1999; 19: 1910-1918Google Scholar, E. S. S. J. U. Leonard W.J. Mol. Biol. Scholar). The of the N-domain also revealed of N-domain X. R. Vinkemeier U. van Darnell Jr., J.E. Kuriyan J. Sci. 2003; Scholar). we in the Stat a conserved residue on the of the N-domain that was to of in Stat1 and in is in and the Stat1 and Stat5b and of was by and of the was reduced in with we Stat5a protein in In these was by an kinase Stat5a protein and 17John S. Vinkemeier U. Soldaini E. Darnell Jr., J.E. Leonard W.J. Mol. Cell. Biol. 1999; 19: 1910-1918Google and this was used in DNA binding The and of Stat1 or tyrosine in response to of and bound to a single site and to wild as and of we the to form on sites by we used an from the IL-2 response that is in both the and genes for the of the IL-2 and S. Vinkemeier U. Soldaini E. Darnell Jr., J.E. Leonard W.J. Mol. Cell. Biol. 1999; 19: 1910-1918Google Scholar, E. S. S. J. U. Leonard W.J. Mol. Biol. Scholar, P. U. Soldaini E. M. J. Biol. 1997; Scholar) have that binding of to single sites from is an Stat5a and Stat5b as with E. S. S. J. U. Leonard W.J. Mol. Biol. of Stat5b on was with Stat5a In to wild of residue to a of formation as indicated by the inability to bind to the in In to nuclear from we used Stat5a proteins to binding to the which the of the analysis with binding sites was also with and two high sites from the was used in these Stat1 can bind to in both a a single Stat1 dimer as as a with two Stat1 dimers. such for with a of the of both wild and the only with the of the was that dimers of only DNA binding and of we in our analysis of a single canonical site and a site that was unrelated or to sites a was used used in Stat1 at a of for these experiments is also the of the Stat1 18Vinkemeier U. Cohen S.L. Moarefi I. Chait B.T. Kuriyan J. 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Genes Dev. 2003; 17: 1992-2005Google Scholar). was that Stat1 can leave the nucleus only after tyrosine and DNA binding was as the of the DNA binding in nuclear Thus, we nuclear accumulation of the diminished nuclear retention due to the that the on DNA our as we a prolonged accumulation in we a to kinase nuclear accumulation of the also in the presence of This is in to wild a of nuclear accumulation an to the of of Stat1 nuclear accumulation (7Meyer T. Marg A. Lemke P. Wiesner B. Vinkemeier U. Genes Dev. 2003; 17: 1992-2005Google Scholar, T. A. I. van M. Vinkemeier U. EMBO J. Scholar). was that can of the In the of a protein this to the of the from the compartment T. A. I. van M. Vinkemeier U. EMBO J. Scholar). This is in for nuclear accumulation in cells. the nuclear retention accumulation was for as was to from the nucleus after of a to our results the of Stat1 is with the of DNA binding (7Meyer T. Marg A. Lemke P. Wiesner B. Vinkemeier U. Genes Dev. 2003; 17: 1992-2005Google Scholar). analysis to this also for both the and wild Stat1 highly nuclear and the was reduced only by the inactivation of tyrosine the was sensitive to phosphatase as wild results indicated that the prolonged nuclear accumulation of the was both from DNA binding and reduced The thus from the nuclear retention to (7Meyer T. Marg A. Lemke P. Wiesner B. Vinkemeier U. Genes Dev. 2003; 17: 1992-2005Google Scholar). the of nuclear accumulation that was with the DNA binding which both and DNA binding (7Meyer T. Marg A. Lemke P. Wiesner B. Vinkemeier U. Genes Dev. 2003; 17: 1992-2005Google be by the of is in this was the as the of nuclear accumulation after of IFNγ can be with the of Stat we a dephosphorylation to be of the This was recovery of proteins or as by in a The for and the of tyrosine dephosphorylation was in cells. which Stat1 M. C. J. C. T. Darnell Jr., J.E. Stark G.R. Kerr I.M. EMBO J. 1993; or that with IL-2 receptor with Stat1 and the for with IFNγ to Stat1 or IL-2 to this the cytokines and the in the or presence of the kinase is in prolonged tyrosine phosphorylation and which is of a reduced dephosphorylation was also for Stat5a and Stat5b in the IL-2 the of dephosphorylation of wild proteins is that in within of residue also prolonged tyrosine phosphorylation as the phosphotyrosine signal for The of to the the phosphotyrosine signal of wild Stat5a/b within after the sensitive to kinase and showed to the signal was for at 2 was is by such as D. R. Stravopodis D. S. J. Ihle J.N. EMBO J. 19: Scholar). of the kinase at the as the the of and dephosphorylation the N-domain of Stat5a/b to the of and wild the that is by the presence of we the of after of M. Scholar). was a of the of both in the and presence of that the of is the of by the a of the IL-2 pathway is that kinase In these show that and are only also defective tyrosine dephosphorylation in the N-domain directly affected the interaction Stat dimer and we in dephosphorylation with and a nuclear tyrosine phosphatase J. M. M. M. K. Mol. Cell. Biol. Scholar). The dephosphorylation of the Stat1 protein was reduced that this is defective in interactions. Thus, the N-domain responsible for cooperative DNA binding of Stat1 dimers is also with the phosphatase TC45. we to of Stat1 to the in dephosphorylation we an on the interaction of phosphatase and which that the dephosphorylation is to of the in a the of the N-domain on gene transcription was gene used to the of and enhanced tyrosine phosphorylation on gene in the on the genes sites as as or by the Stats by the wild Stat1 a with sites by was used was with the as with the wild Stat This was for Stat1 by of a the or of the gene binding sites for and this a single canonical site A. E. L. van J. Biol. 1994; Scholar, J. Z. Proc. Natl. Acad. Sci. U. S. A. 1994; Scholar). of transcription was in gene with and a in which the sites binding sites S. Vinkemeier U. Soldaini E. Darnell Jr., J.E. Leonard W.J. Mol. Cell. Biol. 1999; 19: 1910-1918Google Scholar). IL-2 of the wild was for both Stat5a and Stat5b the gene activation by IL-2 in the of both the their wild at transcription from the high Thus, these results that only sites sensitive to N-domain interactions. also of genes by the seven genes only was by the N-domain of the showed a diminished in with the and which their IFNγ in the Stat1 The of formation for gene in a was by gene in response to to which a Stat1 of with the formation of a of a and which to an unrelated recognition site response S.A. M. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1995; Scholar). genes by of the gene was highly by and of reduced response by the of was affected by the N-domain The formation of Stat and on DNA results from cooperative DNA binding of Stat dimers that to be conserved the Stat and an residue in of the N-domain to this S. Vinkemeier U. Soldaini E. Darnell Jr., J.E. Leonard W.J. Mol. Cell. Biol. 1999; 19: 1910-1918Google Scholar, 18Vinkemeier U. Cohen S.L. Moarefi I. Chait B.T. Kuriyan J. Darnell Jr., J.E. EMBO J. 1996; 15: 5616-5626Google Scholar, X. Darnell Jr., J.E. J. Biol. Scholar). in indicated that of residue of the domain and an was to mediate Stat oligomerization X. R. Vinkemeier U. van Darnell Jr., J.E. Kuriyan J. Sci. 2003; Scholar). This study was to these data that of a conserved residue in the protein a in formation of and Stat5b DNA binding of the of this the was also for D.H. Nat. Scholar). Thus, on these results we the of as for oligomerization of Stat data with most the of N-domain cooperative DNA binding data in transcription be for the to be the of N-domain dimers with at is required for such as study that the that cooperative DNA binding is also required for tyrosine dephosphorylation of Stat1 and as the oligomerization to tyrosine dephosphorylation in using in dephosphorylation with for Stat1 this be to a of dephosphorylation by the phosphatase TC45. of the Stat1 dephosphorylation was by the different of the N-domain of and Stat1. the role of the N-domain in the inactivation of is the domain in the of D. R. Stravopodis D. S. J. Ihle J.N. EMBO J. 19: Scholar, R. R. S. C. X. Hennighausen L. A. B. Mol. Cell. Biol. 1996; Scholar). is that of activated by the is due to from or phosphatase as with and kinase in dephosphorylation of wild the N-domain both dephosphorylation these This that the prolonged tyrosine phosphorylation after of the COOH terminus and in the presence of is in the of and of defective This is by that Jak kinase inactivation and of the receptor S.J. J. Biol. 1997; Scholar, A. J. Biol. Scholar). The of the phosphatase is as different have indicated different such as and to be in the dephosphorylation of J. M. M. M. K. Mol. Cell. Biol. Scholar, T. Mol. Scholar, Wen R. Yang S. J. T. Wang D. J. Biol. 2003; Scholar, S. S. J. Biol. Scholar). The nuclear accumulation of Stats the presence of the N-domain I. C. J. Biol. 1998; Scholar). of residue in Stat1 and in Stat5a/b nuclear after with cytokines. the of of Stat1 prolonged nuclear accumulation dephosphorylation as as deficiency affected the nuclear accumulation of the striking was the of nuclear accumulation which was also with the kinase the of the role that DNA binding in the nuclear accumulation of Stat1. was that to a nuclear signal within the DNA binding domain was by DNA binding C. EMBO J. 19: Scholar). to this DNA binding the nuclear signal and thus nuclear of DNA binding in of nuclear accumulation. this is the retention of DNA binding can be by of with phosphatase (7Meyer T. Marg A. Lemke P. Wiesner B. Vinkemeier U. Genes Dev. 2003; 17: 1992-2005Google or as is in by of the of Stat1 that results the that Stat1 is in the nucleus (7Meyer T. Marg A. Lemke P. Wiesner B. Vinkemeier U. Genes Dev. 2003; 17: 1992-2005Google which dephosphorylation the in the of nuclear this is and DNA that the region required for Stat1 nuclear this region is in the DNA binding domain Thus, DNA binding is for nuclear retention of the DNA bound is to phosphatase the also the concomitant of the nuclear which we the wild and Stat1 found only The of which tyrosine phosphatase results in nuclear of Stat1 dimers. is to that oligomerization of Stat1 is in this due to the form and thus a high in the of dephosphorylation high a that this protein from which also prolonged nuclear retention (7Meyer T. Marg A. Lemke P. Wiesner B. Vinkemeier U. Genes Dev. 2003; 17: 1992-2005Google the dephosphorylation of nuclear Thus, of dephosphorylation was associated with diminished the a nuclear was with transcription have indicated that of gene in a promoter-specific manner. from promoters was with the Stat1 and reduced transcription was used in this of results indicated that the Stats are transcription factors that analysis of or genes in their reduced transcriptional of the Stat1 the these phenotypes are the gene results with promoters for an of sites are by a DNA as with sites. binding to such sites is U. Cohen S.L. Moarefi I. Chait B.T. Kuriyan J. Darnell Jr., J.E. EMBO J. 1996; 15: 5616-5626Google Scholar). The of sites may the that used sites and thus required Stat oligomerization for transcriptional that the only a single canonical site a site also an with only a single discernable such as S.H. K. Mol. Cell. Biol. 1993; was to the of the that binding of the to single or sites was from wild This may indicate that Stat are for transcriptional at promoters that only a single canonical This is by the DNA binding data in and I. the to form on DNA multiple is that of Stat1 are also bound at promoters that only a single canonical This may be to Stat1. In binding site revealed that binding of also can be with a wide range of which in Stat5a binding as a dimer E. S. S. J. U. Leonard W.J. Mol. Biol. Scholar). was that the N-domain is for high DNA binding U. Cohen S.L. Moarefi I. Chait B.T. Kuriyan J. Darnell Jr., J.E. EMBO J. 1996; 15: 5616-5626Google Scholar, 19Xu X. Sun Y.L. T. Science. 1996; and and show that of or in Stat1 or of the dimers with of residue of Stat1 or of was on gene from promoters multiple an for the reduced transcription from promoters be the N-domain a role also in the of transcriptional Vinkemeier U. D. Horvath C.M. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: Scholar). we cannot the that the of residue also of unrelated transcription factors. analysis is required to this In the the that are to by the tyrosine phosphorylation and DNA binding of the Stat The of Stats to with the phosphatase In the activated in of the phosphatase DNA binding the Stat1 protein is from dephosphorylation (7Meyer T. Marg A. Lemke P. Wiesner B. Vinkemeier U. Genes Dev. 2003; 17: 1992-2005Google Scholar). data with to the of the N-domain in and Stats are is to the various in this and I. C. J. Biol. 1998; Scholar, D.H. Nat. Scholar, Vinkemeier U. D. Horvath C.M. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: Scholar) that the of the Stat N-domain a of and high the of to be T. van and and for