Hritish lournal of Haernatologg. 199 1 , 79, Suppl. 1, 9-1 3
Signal transduction pathway activating interferon-alpha-stimulated gene expression SUSANA. VEALS,DANIELS. KESSLER,SERENEJOSIAH, DEBRAG. B. LEONARDA N D DAVIDE. LEVY
Department of Pathology and Kaplan Cancer Center, New York University School of Medicine, New York, U.S.A.
Summary. Interferon-alpha (IFNa) causes profound physiological changes following binding to susceptible target cells. These changes, which include induction of an antiviral state, inhibition of cellular proliferation, and modulation of differentiation, require the transcriptional activation of a set of genes. We have characterized the macromolecular components required for this stimulation of gene expression in order to define the biochemical mechanism of IFNa signal transduction. IFNa stimulated genes (ISGs) are immediate response genes which utilize a pre-existing set of proteins to mediate their induction. A 1 5 bp IFNa-inducible enhancer element present in the promoters of IFNa-stimulated genes,
termed the IFNa stimulated response element (ISRE), is the genetic target for activation of ISGs. This DNA sequence is both necessary and sufficient for transcriptional activation and is the target for action of a positive transcription factor termed ISGF3. The active, DNA-binding form of ISGF3 is only found in cells which have been exposed to IFNa; however, it is activated from a silent form present in all responsive cells. ISGF3 is a multimeric complex assembled from cytoplasmic precursors which are translocated to the nucleus in response to IFNa. Assembly and translocation of ISGF3 is the earliest defined event in the IFNa response pathway.
The action of cytokines to alter the physiology of mammalian cells requires transmission of a signal originating at the cell surface to the cell nucleus to bring about changes in gene expression. One of the unanswered questions in this or any similar signalling process is what is the biochemical nature of the transduced signal which faithfully transmits the immediacy, the magnitude, and the specificity of the inducing ligand. IFNa binds a unique cell-surface receptor to cause profound changes in the metabolism and physiology of target cells. Generation of the antiviral state, cessation of cellular proliferation. and modulation of the differentiation and of the immune status of the cell are all preceded by activation of a discrete set of genes at the transcriptional level. In order to understand the biochemical mechanism leading to the activation of these genes, we have been studying the transcriptional controls and signalling mechanisms responsive to IFNa.
the response pathway. Although the specific antiviral or antiproliferative functions of only a few inducible genes have been defined (Revel & Chebath. 1986). we and others have investigated the molecular events required for induced gene expression (Levyet a/. 1986. 1988, 1989:Kessleret al. 1990: Porter et a/. 1988: Rutherford et al, 1988: Cohen et al, 1988). The rationale for our experiments has been that a definition of nuclear events required for gene expression will lead to elucidation of the more receptor-proximal events of the signalling pathway. Several cDNA clones have been obtained complementary to mRNA molecules whose abundance dramatically increases following treatment of cells with IFNa. Expression studies using these cloned probes established transcriptional mechanisms as the primary basis for this increased abundance. Two features of this induction are relevant to understanding this mechanism. First, the transcriptional response is rapid and proceeds even in the absence of ongoing protein synthesis (e.g. following cycloheximide treatment). This finding demonstrated that all the protein components required for transcriptional induction pre-existed in untreated cells, presumably in a latent state susceptible to activation. Second, although transcriptional activation is rapid, it is also transient, with high levels of transcription produced following IFNcl treatment declining back to near pretreatment levels within a few hours. Unlike the initial activation of transcription, however, this decline in transcription required ongoing cellular protein synthesis. Therefore,
RESULTS
Cis-and trans-acting regulation of lSGs The dependence of the antiviral and antiproliferative actions of IFN on new protein synthesis and induced gene expression defines transcriptional activation as an essential early step in Correspondence:Dr D. E. I ~ v yDepartment . of Pathology and Kaplan Cancer Center. New York University School of Medicine, 5 50 First Avenue, New York. NY 10016. U.S.A.
9
10
Susan A. Veals et al
although there are pre-existing proteins which must become activated following IFNa treatment to produce high rates of transcription, there are new proteins produced which act to return the system to homeostasis (Friedman et al, 1984; Larner et al. 1986). Recombinant expression constructs containing ISG promoters linked to artificial reporter genes have been used to dissect the genetic components necessary for transcriptional induction. A single, IFNa-stimulated response element (ISRE) in the 5'-flanking region of ISGs was found to be necessary and sufficient for IFNa-stimulated transcriptional responses (Levy et al. 1988; Reich 81Darnell. 1989; Cohen et al, 1988: Porter et al. 1988). This 1 5 bp DNA sequence acted as a n IFNa-stimulated enhancer, functioning in either orientation and at different distances from the start of transcription. Extensive point mutagenesis of this element has defined the precise nucleotides required for the response (Kessler et ul. 1988). Enhancer action is generally mediated through the binding of sequence-specific nuclear proteins. Several protein factors have been defined which specifically recognize the ISRE. some of which are unique to IFNa-treated cells. Of these different ISRE-binding factors, a single transcription factor, termed ISGF3, correlates with the positive transactivation of ISG transcription (Levy et al. 1988, 1989; Kessler et al. 1988, 1990). The activity of this factor is detected only following IFNr treatment, but it is composed from pre-existing polypeptides and its activation, like transcription, is not inhibited by cycloheximide. Its sequence requirements for binding at the ISRE correlate precisely with the requirements for transcriptional activation as defined by mutagenesis, and its kinetics of activation and its absence from IFNa-resistant cell lines also correlate with ISG transcription. In addition, ISGF3 purified away from all other ISRE-binding factors will direct ISREdependent transcriptional initiation in vitro (Fu et al. 1990). Synergy between 1FNa and l F N y through modulation of transcription factor subunits The combined action of type I (IFNa/,!l) and type 11 (IFNy) interferons has been noted to produce synergistic physiological responses greater than the response to either interferon alone. This observation suggests that the two IFNs act through distinct mechanisms, although in some cases through the induction of similar genes (Revel & Chebath. 1986). In our studies on the transcriptional induction of IFNa-stimulated genes, we noted a synergistic effect of pretreatment with IFNy. In responsive cell lines, ISGs are transcriptionally induced to high levels in response to IFNa but are unaffected by treatment with IFNy. However, in some cell types (e.g. HeLa S3 tumour cells, A549 epithelial carcinoma cells, Cos- 1 transformed embryonic kidney cells), although IFNy still does not induce expression of the ISGs. pretreatment of these cells with IFNy showed a profound effect on subsequent IFNa stimulation. Cells pretreated for 18-24 h with IFNy were rendered supersensitive so that subsequent exposure to IFNa produced levels of ISG transcription approximately 10-fold higher than in cells which had not been so treated (Levy et al, 1990). The interaction between IFNa and IFNYto oroduce high ., I
.
rates of ISG transcription resulted from increased levels of ISGF3. Although activation of ISGF3 in response to IFNr treatment does not require protein synthesis. it was found that accumulation of a specific protein was required for the priming effect of IFNy. This protein accumulated to high c
C Q
c
8 E
t U
3 0 I
8 i0
L
8I
Q n 2
a Q
p!
LL I
A G
T T T
C G G T T T
Fig 1. ISGF3y is an ISRE-specific DNA binding factor. Mobility-shift assays were performed using ISGF3 purified from nuclei or formed iri vitro from cytoplasmic ISGF3a and ISGF 3y and using cytoplasmic ISGF3y alone. Labelled DNA in the shift complexes as well as unbound DNA were cleaved in situ by soaking the inact gel in 1 ,lophenanthroline and copper sulphate (Kuwabara & Sigman. 1987). DNA was recovered and fractionated by denaturing gel electrophoresis. as indicated. The sequence of the ISRE is indicated to the right. Both nuclear and cytoplasmic ISGF3 displayed strong protection of the ISRE, while ISGF3y alone displayed a somewhat weaker protection of the same DNA sequence.
1FNu Signal Transduction
11
Fig 2. Glycerol gradient sedimentation fractionates ISGF3 into separate ISGF3a and ISGF3y activities. ISGF3 purified from nuclei of n%-treated HeLa cells (A-C) or ISGF3a (D)and ISCF39 (E) purified from cytoplasm were loaded onto 1 5 3 0 % glycerol gradients and fractionated by centrifugation.Starting material (L) and each fraction were assayed for ISRE-binding activity by mobility-shift either directly (A) or after addition of ISCF37 (Band D) or ISGF3a (C and E). as indicated.Sedimentationof marker proteins was determined by SDS-PAGE of fractions from a parallel gradient as indicated by their molecular weights.
levels in IFNy-treated cells which allowed activation of increased levels of ISGF3 following treatment with IFNa. The protein which was produced in response to IFNy has been found to be a subunit of mature ISGF3 (Kessler ~t aL 1990).
Cytoplasmic activation of ISGF3 Activation of ISCF3 as a nuclear, DNA-binding protein is an early event in the stimulation of gene expression following IFNa binding to its cell surface receptor. The activation of
12
Susan A. Veals et al
latent ISGF3 to a form competent for DNA binding and transcriptional activation has been found to occur in the cytoplasm of IFNa-treated cells and to involve at least three steps. First. an as yet uncharacterized biochemical modification initiates the activation process. This is followed by translocation of the active factor from the cytoplasm to the nucleus where final assembly of the complete, multimeric complex occurs on the ISRE. The cytoplasmic localization of the proteins that interact to form ISGF3 was demonstrated by two types of experiments. First, following IFNa treatment active ISGF3 can be detected immediately in the cytoplasm but only after 5 min in the nucleus (Levy et al. 1989). Second, enucleated cytoplasts can respond to IFNa to produce cytoplasmic activated ISGF3 (Dale et al, 1989). The assembly of multimeric ISGF3 from cytoplasmic components has been reconstituted in vitro and has provided an explanation for the synergy previously observed between IFNy and IFNa. Mixture of cytoplasm from IFNa-treated cells with cytoplasm from untreated cells resulted in formation of additional active ISGF3. demonstrating the presence of excess components. When a similar mixing experiment was performed using extracts from IFNa-treated cells and from IFNytreated cells, very large amounts of active ISGF3 were observed (Levy et al. 1990).This increase in the activation of ISGF3 was due to the presence of large amounts of the subunit of ISGF3 synthesized in response to IFNy. lSGF3 is composed ojjour polypeptides Reconstitution in vitro of cytoplasmic activation of ISGF3 has allowed biochemical characterization of the components of this factor. The component produced in response to IFNy treatment, termed ISGF3y. is a 48 kDa protein displaying ISRE-specific DNA binding activity (Fig 1). This protein has been purified to homogeneity: partial protein sequence derived from tryptic peptides has shown no significant homology with known proteins. Intact ISGF3 can be separated into two components (Fig 2). one of which is the 48 kDa ISGF3y. the other being composed of at least three additional polypeptides. This second component, termed ISGF3a, was detected only in cells which had been treated with IFNa and may be the direct target for the protein modification event activated by the IFNa receptor. The three polypeptides comprising this component cofractionated during purification, interacted with ISGF3y to form the mature. ISREbinding factor, but displayed no intrinsic DNA binding activity themselves. However, the interaction between ISGF3a and ISGF3y stabilized the binding of ISGF3y on the ISRE by increasing the affinity of binding approximately 25-fold (see Fig 1). The stimulation of active, DNA-binding ISGF3 in response to IFNa treatment is a result of immediate cytoplasmic activation of the ISGF3a component. Although the biochemical event facilitating this activation has not yet been characterized, it appears that phosphorylation of ISGF3a is required for activity. First, treatment of ISGF3 with phosphatases in vitro has been found to reduce their ability to bind DNA. Second, kinase inhibitors in vivo can prevent the activation of ISGF3 in response to IFNa. Two potent inhibitors of Ca2+/phospholipid-dependentprotein kinases, staur-
osporine and its derivative K-2 52a. prevented the appearance of active ISGF3 following IFNa treatment. Specifically. the presence of these inhibitors prevented the activation of the ISGF3a component. However, two other inhibitors of similar kinases. H-7 and H-8,had only a minimal effect on ISGF3 activation (Lew et al. 1989).
DISCUSSION Several of the macromolecular events leading to the activation of gene expression following IFNa treatment of responsive cells have been defined. Responsive genes contain a promoter-proximal element, the ISRE. which acts as an IFNa-stimulatedenhancer element. The presence of this DNA sequence in a series of distinct genes directs their coordinate activation in response to a specific cell-surface signal. The activity of the ISRE is dependent on proteins which recognize it in a sequence-specific manner. The initial activation of ISRE-directed transcription requires assembly of the multimeric factor ISGF3 on this DNA element. ISGF3 is activated from latent components resident in the cytoplasm of IFNa-responsive cells. One of these components. the 48 kDa ISGF3y. is the DNA-recognition element of ISGF 3 . It binds the ISRE in a sequence-specificmanner, but displays a relatively low affinity for its target sequence. The second component, ISGF3a, which is composed of three polypeptides, associates with ISGF3y to stabilize its binding on the ISRE by increasing the affinity of the interaction. While ISGF3y is induced to high abundance in response to IFNy treatment and is thus responsible for the observed transcriptional synergy between the two types of IFN, ISGF30: is the initial target of the IFNa signal response pathway. This component of ISGF3 is modified in IFNa-treated cells to facilitate nuclear translocation and final factor assembly. The signal transduction pathway initiated by IFNa binding to its cell surface receptor may be a model for how different cell surface ligands can activate discrete sets of genes in a single cell type. There is clearly a high degree of specificity inherent in a ligand-receptor interaction, and this information must be accurately relayed to the nucleus. Although it has been postulated that small-molecule second messengers carry such information, it seems that in the IFNa system, and possibly other ligand-receptor systems as well (Lenardo et al. 1989), signal transduction is mediated by biospecific interactions of macromolecules. Thus, the specific recognition of IFNa by its receptor causes a modification of a cytoplasmic protein, enabling this protein to translocate to the nucleus. interact with additional polypeptides, and assemble an active transcription complex on DNA. The biochemical nature of the initial modification reaction remains to be determined as does the role of phosphorylation in the activity of the mature transcription factor. Whether the kinases which were inhibited by staurosporine and K-252a play a regulatory as opposed to a maintenance role in ISGF3 activity, we would predict that any phosphorylation events in the direct signal transduction pathway act in a highly specific, localized manner rather than through a global activation of a general kinase such as protein kinase C.
1FNa Signal Transduction REFERENCES Cohen. B.. Peretz. D.. Vaiman. D..Benech. P. & Chebath. j. (1988) Enhancer-like interferon responsive sequences of the human and murine ( 2 ’ 4 ) oligoadenylate synthetase gene promoters. The EMBO/ournal. 7, 1411-1419. Dale. T.C.. Imam, A.M.A.. Kerr. I.M. & Stark, G.R. (1989) Rapid activation by interferon a of a latent DNA-binding protein present in the cytoplasm of untreated cells. Proceedings of the National Academy ojSciences oJthe UnitedStates of America. 86, 1203-1 207. Friedman. R.L.. Manly. S.P.. McMahon. M.. Kerr. I.M. & Stark. G.R. ( 1 984) Transcriptional and post-transcriptional regulation of interferon-induced gene expression in human cells. Cell, 3 8 , 745755. Fu. X.-Y.. Kessler. D.S.. Veals. S.A.. Levy. D.E. & Darnell, J.E.. Jr (1 990) ISGF3. the transcriptional activator induced by interferon a, consists of multiple interacting polypeptide chains. Proceedings o/ the National Academg oJScienres ofthe United Stafes o/ America, 87, 8 5 5 5-8 5 59. Kessler. I).S.. Levy. D.E. & Darnell. J.E., J r (1988) Two interferoninduced nuclear factors bind a single promoter element in interferon-stimulated genes. Proceedings oJthe National Aradem!l oJ Scienres ofthe United States ofAmerica, 8 5 . 8521-8525. Kessler. D.S.. Veals. S.A.. Fu. X.-Y. & Levy. D.E. (1990) IFN-alpha regulates nuclear translocation and DNA-binding affinity of ISGF 3 , a multimeric transcriptional activator. Genes and Lkvdopment. 4, 1753-1 765. Kuwabara. M.D. & Sigman, D.S. ( 1 987) Footprinting DNA-protein complexes in situ following gel retardation using I , 1O-phenanthroline-copper ion: Escherichia coli RNA polymerase-lac promoter complexes. Biochemistry, 26. 72 34-7238. Lamer. A.C.. Chaudhuri. A. & Darnell. J.E..Jr ( 1 986)Transcriptional induction by interferon. lournal of Biological Chemistry. 261,45 3459. Lenardo. M.J.. Fan. C.M.. Maniatis. T. & Baltimore, D. (1989) The involvement of NF-kB in 8-interferon gene regulation reveals its
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role as a widely inducible mediator of signal transduction. Cell. 57, 287-294. Levy, D.E.. Kessler. D.S., Pine, R. & Darnell. J.E.. Jr (1989) Cytoplasmic activation of ISGF3. the positive regulator of interferon-alphastimulated transcription. reconstituted in vitro. Genes and Development. 3 , 1362-1371. Levy, D.E.. Kessler. D.S.. Pine. R.. Reich. N. & Darnell, J.E., J r (1988) Interferon-induced nuclear factors that bind a shared promoter element correlate with positive and negative control. Genes and Deiwlopment. 2. 383-393. Levy. D.E.. Lamer, A.C.. Chaudhuri. A.. Babis. L.E. & Darnell. J.E..Jr ( 1986) Interferon-stimulated transcription: isolation of an inducible gene and identification of its regulatory region. Proceedings oJ the National Academy ofSciences oJthe United States of America. 8 3 . 8929-8933. Levy.D.E.. Lew. D.J.. Decker. T.. Kessler. D.S. & Darnell. J.E.. Jr ( 1 990) Synergistic interaction between interferon-a: and interferon-y through induced synthesis of one subunit of the transcription factor ISGF3. The EMBO Journal. 9, 1105-1 1 1 1 . Lew. D.J.. Decker. T. & Darnell. J.E.. Jr (1989) Alpha interferon and gamma interferon stimulate transcription of a single gene through different signal transduction pathways. Molecular and Cellular Biology. 9, 5404-541 1 . Porter, A.C.G.. Chernajovsky. Y.. Dale, T.C.. Gilbert. C.S.. Stark, G.R. & Kerr. I.M. (1988) Interferon response element of the human gene 6-16. The EMBOIournal. 7, 85-92. Reich. N. & Darnell. J.E.. Jr ( 1 989) Differential binding of interferoninduced factors to an oligonucleotide that mediates transcriptional activation. Nucleic Acids Research, 17, 341 5-3424. Revel. M. & Chebath. J. (1986)Interferon-activated genes. Trends in Biochemical Sciences. 11, 166-1 70. Rutherford. M.N.. Hannigan. G.E. & Williams. B.R.G. (1988) Interferon-induced binding of nuclear factors to promoter elements of the 2-5A synthetase gene. The EMBO journal. 7, 751-759.
Signal transduction pathway activating interferon-alpha-stimulated gene expression SUSANA. VEALS,DANIELS. KESSLER,SERENEJOSIAH, DEBRAG. B. LEONARDA N D DAVIDE. LEVY
Department of Pathology and Kaplan Cancer Center, New York University School of Medicine, New York, U.S.A.
Summary. Interferon-alpha (IFNa) causes profound physiological changes following binding to susceptible target cells. These changes, which include induction of an antiviral state, inhibition of cellular proliferation, and modulation of differentiation, require the transcriptional activation of a set of genes. We have characterized the macromolecular components required for this stimulation of gene expression in order to define the biochemical mechanism of IFNa signal transduction. IFNa stimulated genes (ISGs) are immediate response genes which utilize a pre-existing set of proteins to mediate their induction. A 1 5 bp IFNa-inducible enhancer element present in the promoters of IFNa-stimulated genes,
termed the IFNa stimulated response element (ISRE), is the genetic target for activation of ISGs. This DNA sequence is both necessary and sufficient for transcriptional activation and is the target for action of a positive transcription factor termed ISGF3. The active, DNA-binding form of ISGF3 is only found in cells which have been exposed to IFNa; however, it is activated from a silent form present in all responsive cells. ISGF3 is a multimeric complex assembled from cytoplasmic precursors which are translocated to the nucleus in response to IFNa. Assembly and translocation of ISGF3 is the earliest defined event in the IFNa response pathway.
The action of cytokines to alter the physiology of mammalian cells requires transmission of a signal originating at the cell surface to the cell nucleus to bring about changes in gene expression. One of the unanswered questions in this or any similar signalling process is what is the biochemical nature of the transduced signal which faithfully transmits the immediacy, the magnitude, and the specificity of the inducing ligand. IFNa binds a unique cell-surface receptor to cause profound changes in the metabolism and physiology of target cells. Generation of the antiviral state, cessation of cellular proliferation. and modulation of the differentiation and of the immune status of the cell are all preceded by activation of a discrete set of genes at the transcriptional level. In order to understand the biochemical mechanism leading to the activation of these genes, we have been studying the transcriptional controls and signalling mechanisms responsive to IFNa.
the response pathway. Although the specific antiviral or antiproliferative functions of only a few inducible genes have been defined (Revel & Chebath. 1986). we and others have investigated the molecular events required for induced gene expression (Levyet a/. 1986. 1988, 1989:Kessleret al. 1990: Porter et a/. 1988: Rutherford et al, 1988: Cohen et al, 1988). The rationale for our experiments has been that a definition of nuclear events required for gene expression will lead to elucidation of the more receptor-proximal events of the signalling pathway. Several cDNA clones have been obtained complementary to mRNA molecules whose abundance dramatically increases following treatment of cells with IFNa. Expression studies using these cloned probes established transcriptional mechanisms as the primary basis for this increased abundance. Two features of this induction are relevant to understanding this mechanism. First, the transcriptional response is rapid and proceeds even in the absence of ongoing protein synthesis (e.g. following cycloheximide treatment). This finding demonstrated that all the protein components required for transcriptional induction pre-existed in untreated cells, presumably in a latent state susceptible to activation. Second, although transcriptional activation is rapid, it is also transient, with high levels of transcription produced following IFNcl treatment declining back to near pretreatment levels within a few hours. Unlike the initial activation of transcription, however, this decline in transcription required ongoing cellular protein synthesis. Therefore,
RESULTS
Cis-and trans-acting regulation of lSGs The dependence of the antiviral and antiproliferative actions of IFN on new protein synthesis and induced gene expression defines transcriptional activation as an essential early step in Correspondence:Dr D. E. I ~ v yDepartment . of Pathology and Kaplan Cancer Center. New York University School of Medicine, 5 50 First Avenue, New York. NY 10016. U.S.A.
9
10
Susan A. Veals et al
although there are pre-existing proteins which must become activated following IFNa treatment to produce high rates of transcription, there are new proteins produced which act to return the system to homeostasis (Friedman et al, 1984; Larner et al. 1986). Recombinant expression constructs containing ISG promoters linked to artificial reporter genes have been used to dissect the genetic components necessary for transcriptional induction. A single, IFNa-stimulated response element (ISRE) in the 5'-flanking region of ISGs was found to be necessary and sufficient for IFNa-stimulated transcriptional responses (Levy et al. 1988; Reich 81Darnell. 1989; Cohen et al, 1988: Porter et al. 1988). This 1 5 bp DNA sequence acted as a n IFNa-stimulated enhancer, functioning in either orientation and at different distances from the start of transcription. Extensive point mutagenesis of this element has defined the precise nucleotides required for the response (Kessler et ul. 1988). Enhancer action is generally mediated through the binding of sequence-specific nuclear proteins. Several protein factors have been defined which specifically recognize the ISRE. some of which are unique to IFNa-treated cells. Of these different ISRE-binding factors, a single transcription factor, termed ISGF3, correlates with the positive transactivation of ISG transcription (Levy et al. 1988, 1989; Kessler et al. 1988, 1990). The activity of this factor is detected only following IFNr treatment, but it is composed from pre-existing polypeptides and its activation, like transcription, is not inhibited by cycloheximide. Its sequence requirements for binding at the ISRE correlate precisely with the requirements for transcriptional activation as defined by mutagenesis, and its kinetics of activation and its absence from IFNa-resistant cell lines also correlate with ISG transcription. In addition, ISGF3 purified away from all other ISRE-binding factors will direct ISREdependent transcriptional initiation in vitro (Fu et al. 1990). Synergy between 1FNa and l F N y through modulation of transcription factor subunits The combined action of type I (IFNa/,!l) and type 11 (IFNy) interferons has been noted to produce synergistic physiological responses greater than the response to either interferon alone. This observation suggests that the two IFNs act through distinct mechanisms, although in some cases through the induction of similar genes (Revel & Chebath. 1986). In our studies on the transcriptional induction of IFNa-stimulated genes, we noted a synergistic effect of pretreatment with IFNy. In responsive cell lines, ISGs are transcriptionally induced to high levels in response to IFNa but are unaffected by treatment with IFNy. However, in some cell types (e.g. HeLa S3 tumour cells, A549 epithelial carcinoma cells, Cos- 1 transformed embryonic kidney cells), although IFNy still does not induce expression of the ISGs. pretreatment of these cells with IFNy showed a profound effect on subsequent IFNa stimulation. Cells pretreated for 18-24 h with IFNy were rendered supersensitive so that subsequent exposure to IFNa produced levels of ISG transcription approximately 10-fold higher than in cells which had not been so treated (Levy et al, 1990). The interaction between IFNa and IFNYto oroduce high ., I
.
rates of ISG transcription resulted from increased levels of ISGF3. Although activation of ISGF3 in response to IFNr treatment does not require protein synthesis. it was found that accumulation of a specific protein was required for the priming effect of IFNy. This protein accumulated to high c
C Q
c
8 E
t U
3 0 I
8 i0
L
8I
Q n 2
a Q
p!
LL I
A G
T T T
C G G T T T
Fig 1. ISGF3y is an ISRE-specific DNA binding factor. Mobility-shift assays were performed using ISGF3 purified from nuclei or formed iri vitro from cytoplasmic ISGF3a and ISGF 3y and using cytoplasmic ISGF3y alone. Labelled DNA in the shift complexes as well as unbound DNA were cleaved in situ by soaking the inact gel in 1 ,lophenanthroline and copper sulphate (Kuwabara & Sigman. 1987). DNA was recovered and fractionated by denaturing gel electrophoresis. as indicated. The sequence of the ISRE is indicated to the right. Both nuclear and cytoplasmic ISGF3 displayed strong protection of the ISRE, while ISGF3y alone displayed a somewhat weaker protection of the same DNA sequence.
1FNu Signal Transduction
11
Fig 2. Glycerol gradient sedimentation fractionates ISGF3 into separate ISGF3a and ISGF3y activities. ISGF3 purified from nuclei of n%-treated HeLa cells (A-C) or ISGF3a (D)and ISCF39 (E) purified from cytoplasm were loaded onto 1 5 3 0 % glycerol gradients and fractionated by centrifugation.Starting material (L) and each fraction were assayed for ISRE-binding activity by mobility-shift either directly (A) or after addition of ISCF37 (Band D) or ISGF3a (C and E). as indicated.Sedimentationof marker proteins was determined by SDS-PAGE of fractions from a parallel gradient as indicated by their molecular weights.
levels in IFNy-treated cells which allowed activation of increased levels of ISGF3 following treatment with IFNa. The protein which was produced in response to IFNy has been found to be a subunit of mature ISGF3 (Kessler ~t aL 1990).
Cytoplasmic activation of ISGF3 Activation of ISCF3 as a nuclear, DNA-binding protein is an early event in the stimulation of gene expression following IFNa binding to its cell surface receptor. The activation of
12
Susan A. Veals et al
latent ISGF3 to a form competent for DNA binding and transcriptional activation has been found to occur in the cytoplasm of IFNa-treated cells and to involve at least three steps. First. an as yet uncharacterized biochemical modification initiates the activation process. This is followed by translocation of the active factor from the cytoplasm to the nucleus where final assembly of the complete, multimeric complex occurs on the ISRE. The cytoplasmic localization of the proteins that interact to form ISGF3 was demonstrated by two types of experiments. First, following IFNa treatment active ISGF3 can be detected immediately in the cytoplasm but only after 5 min in the nucleus (Levy et al. 1989). Second, enucleated cytoplasts can respond to IFNa to produce cytoplasmic activated ISGF3 (Dale et al, 1989). The assembly of multimeric ISGF3 from cytoplasmic components has been reconstituted in vitro and has provided an explanation for the synergy previously observed between IFNy and IFNa. Mixture of cytoplasm from IFNa-treated cells with cytoplasm from untreated cells resulted in formation of additional active ISGF3. demonstrating the presence of excess components. When a similar mixing experiment was performed using extracts from IFNa-treated cells and from IFNytreated cells, very large amounts of active ISGF3 were observed (Levy et al. 1990).This increase in the activation of ISGF3 was due to the presence of large amounts of the subunit of ISGF3 synthesized in response to IFNy. lSGF3 is composed ojjour polypeptides Reconstitution in vitro of cytoplasmic activation of ISGF3 has allowed biochemical characterization of the components of this factor. The component produced in response to IFNy treatment, termed ISGF3y. is a 48 kDa protein displaying ISRE-specific DNA binding activity (Fig 1). This protein has been purified to homogeneity: partial protein sequence derived from tryptic peptides has shown no significant homology with known proteins. Intact ISGF3 can be separated into two components (Fig 2). one of which is the 48 kDa ISGF3y. the other being composed of at least three additional polypeptides. This second component, termed ISGF3a, was detected only in cells which had been treated with IFNa and may be the direct target for the protein modification event activated by the IFNa receptor. The three polypeptides comprising this component cofractionated during purification, interacted with ISGF3y to form the mature. ISREbinding factor, but displayed no intrinsic DNA binding activity themselves. However, the interaction between ISGF3a and ISGF3y stabilized the binding of ISGF3y on the ISRE by increasing the affinity of binding approximately 25-fold (see Fig 1). The stimulation of active, DNA-binding ISGF3 in response to IFNa treatment is a result of immediate cytoplasmic activation of the ISGF3a component. Although the biochemical event facilitating this activation has not yet been characterized, it appears that phosphorylation of ISGF3a is required for activity. First, treatment of ISGF3 with phosphatases in vitro has been found to reduce their ability to bind DNA. Second, kinase inhibitors in vivo can prevent the activation of ISGF3 in response to IFNa. Two potent inhibitors of Ca2+/phospholipid-dependentprotein kinases, staur-
osporine and its derivative K-2 52a. prevented the appearance of active ISGF3 following IFNa treatment. Specifically. the presence of these inhibitors prevented the activation of the ISGF3a component. However, two other inhibitors of similar kinases. H-7 and H-8,had only a minimal effect on ISGF3 activation (Lew et al. 1989).
DISCUSSION Several of the macromolecular events leading to the activation of gene expression following IFNa treatment of responsive cells have been defined. Responsive genes contain a promoter-proximal element, the ISRE. which acts as an IFNa-stimulatedenhancer element. The presence of this DNA sequence in a series of distinct genes directs their coordinate activation in response to a specific cell-surface signal. The activity of the ISRE is dependent on proteins which recognize it in a sequence-specific manner. The initial activation of ISRE-directed transcription requires assembly of the multimeric factor ISGF3 on this DNA element. ISGF3 is activated from latent components resident in the cytoplasm of IFNa-responsive cells. One of these components. the 48 kDa ISGF3y. is the DNA-recognition element of ISGF 3 . It binds the ISRE in a sequence-specificmanner, but displays a relatively low affinity for its target sequence. The second component, ISGF3a, which is composed of three polypeptides, associates with ISGF3y to stabilize its binding on the ISRE by increasing the affinity of the interaction. While ISGF3y is induced to high abundance in response to IFNy treatment and is thus responsible for the observed transcriptional synergy between the two types of IFN, ISGF30: is the initial target of the IFNa signal response pathway. This component of ISGF3 is modified in IFNa-treated cells to facilitate nuclear translocation and final factor assembly. The signal transduction pathway initiated by IFNa binding to its cell surface receptor may be a model for how different cell surface ligands can activate discrete sets of genes in a single cell type. There is clearly a high degree of specificity inherent in a ligand-receptor interaction, and this information must be accurately relayed to the nucleus. Although it has been postulated that small-molecule second messengers carry such information, it seems that in the IFNa system, and possibly other ligand-receptor systems as well (Lenardo et al. 1989), signal transduction is mediated by biospecific interactions of macromolecules. Thus, the specific recognition of IFNa by its receptor causes a modification of a cytoplasmic protein, enabling this protein to translocate to the nucleus. interact with additional polypeptides, and assemble an active transcription complex on DNA. The biochemical nature of the initial modification reaction remains to be determined as does the role of phosphorylation in the activity of the mature transcription factor. Whether the kinases which were inhibited by staurosporine and K-252a play a regulatory as opposed to a maintenance role in ISGF3 activity, we would predict that any phosphorylation events in the direct signal transduction pathway act in a highly specific, localized manner rather than through a global activation of a general kinase such as protein kinase C.
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