and gene control
Extracellular signals regulate gene expression by triggering signal transduction cascades that result in the modulation of transcription factor activity. This is most commonly achieved by changes in the phosphorylation state of these nuclear proteins. Phosphorylation affects transcription factor activity at several distinct levels. It can modulate their intracellular localization by controlling the association with other proteins, have both negative and positive effects on their DNA-binding activity, and modulate the activity of their transcriptional activation domains. In addition to phosphorylation, protein-protein interactions also have an important role in mediating a crosstalk at the nuclear level between different signalling pathways. Current
in Cell Biology
The ability to respond to extracellular signals is essential for the development, survival, and the potential of all living organisms to adapt to changing and adverse environmental conditions A common response to extracellular signals involves changes in the spectrum and rates of gene expression. In prokaryotes and unicellular eukaryotes, some of these responses cause the differentiation of the ceU into a form (spore) that is much less susceptible to environmental damage. Other signals allow unicellular organisms to adapt to new nutritional conditions by repressing the expression of unnecessary metabolic and biosynthetic enzymes and by inducing the expression of others. Such protective and adaptive responses have probably served as evolutionary precursors to the more complex differentiation programs of multiceUular organisms, most of which are also controlled by extracellular signals. Multicellular organisms have also developed the capacity to generate many different internal signals in the forms of hormones, growth factors and morphogens, and to use them for maintaining homeostasis and adaptation to new nutritional requirements and to control ceU differentiation and proliferation. One line of evidence for these evolutionary relationships is the high degree of sequence conservation among transcriptional regulators such as GCN4 and c-Jun or HAP213 and CBP. The most common way to regulate gene expression in response to extracellular signals, is by modulating the activity of transcription factors that recognize specific c&acting elements in the control regions of the genes, the transcription of which is affected by these signals. In bacteria, the transduction of extracellular signals to the transcriptional machinery, usually occurs by a two-component sys-
CRE-cAMP GSR-glycogen M-protein
tem. One component serves as a sensor for the extracellular signal and transmits this information via protein phosphorylation to a second component that functions as a transcriptional activator [ 11. Because of the increased physical distance and separation between cell-surface receptors and the nuclear transcriptional machinery, eukaryotic cells have evolved far more intricate pathways for signal transduction; however, as outlined later, the basic operating principle is the same as in prokaryotes: an extracellular signal affects the activity of a proteinkinase cascade that modulates transcription-factor activity by protein phosphorylation. During the past 30 years, most of the activity in the field of signal transduction has concentrated on the identification of hormones and growth factors, receptors for these molecules, second messengers, enzymes that generate second messengers and the mechanisms that control their activity. Only in the past 5 years has it been feasible to approach what appears to be the final step in signal transduction, the control of transcription factor activity An important step in this progression was the identification of c&responsive elements that are responsible for modulation of gene expression in response to various extracellular signals. With the recent identification and cloning of several transcription factors that interact with such elements, it has been possible to embark on the biochemical analysis of the mechanisms by which signalling pathways control the activity of these transcriptional regulators. While this field is still very much in its infancy, studies on several mammalian transcription factors demonstrate some of the basic biochemical mechanisms that are used to control their activity. Rather than give an exhaustive survey of the literature, I will use a few selected examples to illustrate what has been learned in
Abbreviations response element; CREB-cAMP-response-element-binding
protein; CCR-glucocorticoid receptor; synthase kinase; HRE-hormone response element; W-interferon; Ilk-interferon regulatory factor; kinase A; WCS-protein kinase C; SE--serum response element; -rum response factor; TPA-12-O-tetradecanoyl-phorbol-13-acetate; TRE-TPA response element. @ Current
Ltd ISSN 0955*74
Nucleus and gene expression the past 18 months on the control of transcription factor activity by signal transduction pathways.
CREB DNA-binding activity , these findings have not been confirmed, and regulation of CREB activity appears to involve only its activation domain.
CAMP-response-element-binding the response to CAMP
According to a recent report, CREB activity is induced not only by CAMP but also by elevated intracellular Ca*+ [8*]. A ck element responsible for induction of clfos in response to elevated Ca2+ has been mapped and found to be indistinguishable from the CRE when tested in PC12 pheochromocytoma cells. In tttro, this element is recognized by CREB and, in response to stimuli which elevate intracellular CaZ+ in PC12 cells, CREB is phosphotylated on a site identical to the one phosphotylated by PKA [8-l. These results are intriguing because Ca’+ does not activate PKA but a variety of other protein kinases such as the CaM kinase and, in most cells, CREcontaining genes are not induced in response to Ca2+. It is possible, however, that this convergence of the CAMP and Ca2+ path ways is unique to PC12 cells (and maybe other neuronal cell types) but does not apply to other cell types.
Cyclic AMP-response-element-binding protein (CREB) is a 43 kD DNA-binding protein that is responsible for transcriptional activation of many cAMP-inducible genes. It recognizes a specific response element, the cAh4P response Ielement (WE), in the control regions of CAMPinducible genes to bring about their transcriptional activation [ 2,3]. Cyclic AMP, the first second messenger to be identikd, is generated by adenylate cyclase in response to stimulation of various cell-surface receptors coupled to GTP-binding proteins. It binds to the regulatory subunit of protein kinase A (PKA) to elicit its dissociation from the catalytic subunit. The free and active catalytic subunit translocates to the nucleus , where it is thought to phosphory late CREB and increase its ability to activate transcription [5-l. The CEEB open reading frame was found to contain a consensus PKA phosphotylation site [ 2,3]. In z’iza 32P labelling experiments have confirmed that phosphorylation of this site is stimulated in response to treatment of cells with agents that elevate the intracellular level of CAMP. The same site is phosphorylated in vitro by purified PKA [ 5**]. Interestingly, the PKA-regulated phosphotylation site was shown to reside within the CREB activation domain [5**,6]. Inactivation of this site by targeted muta genesis indicates that its phosphorylation is critical for stimulation of the CKEB transactivation function by PKA [5-l. Although th e exact mechanism by which phosphorylation at this site increases the ability of CREB to activate transcription is not known, it does not involve an increase in the negative charge of the activation domain because replacement of the phosphoryiatable serine residue by a negatively charged residue does not activate the protein [5**]. A more probable mechanism may involve a phosphorylation-induced conformational change of the CREB activation domain which increases its ability to interact with a yet to be identified target that is part of the basic transcriptional machinery. Interestingly, alternative splicing generates a variant of the CREB protein, ACEEB, which is more abundant than CREB itself. ACREB does not have a short region, the a-region, which is adjacent to the PKA phosphorylation site. Because of the absence of this region, ACKEB is only one tenth as active as CREB
L7.1. It was suggested that PKA phosphotylation may stimulate CREB activity by modulating the structure of the a-region [7*]. While elucidation of the exact mechanism by which PKA-mediated phosphorylation controls CREB activity depends on determination of the tertiary structure of CREB and identikation of the target with which it interacts, it is clear that CREB is the major nuclear target for the cAh4P-mediated signal transduction pathway. Despite early reports that phosphorylation may also aifect
Not all cAh4P-inducible genes are directly activated by CREB. In the case of the growth-hormone gene, the expression of which is induced in response to elevated intracellular cAh4P in pituitary cells, promoter analysis failed to reveal CREs that could be separated from cell-type specific promoter elements [ 91. These elements are recognized by GHF-1, a pituitaryspeciiic transcription factor that belongs to the homeodomain family [lo]. Analysis of GHF-I expression indicates that it is induced by CAMP and that this induction is mediated by CREB which binds to two sites within the GHFl promoter [ 11.1. Therefore, in this case, induction of a responsive gene by cAMP is mediated via an indirect mechanism that involves increased production of a cell-type-specific transcription factor. It remains to be determined how many of the effects of cAh4P on gene transcription are mediated by a similar mechanism, while the control of expression of various cell-type-specific activators by CAMP can serve to increase the spectrum of genes regulated by this signalling system. AP-1: positive phosphorylation
API is a transcription factor that mediates gene induction by phorbol ester tumor promoters such as 12.O-tetradecanoyl-phorbol-13.acetate (TPA), which are potent activators of protein kinase C (PKC) [ 121. In addition to TPA, AP-1 activity is induced by many polypeptide growth factors, cytokines, and activated oncoproteins [ 131. API is not a unique protein but a collection of homodimeric and heterodomeric complexes composed of the various jun and f~ gene products, all of which interact with a common binding site, the TPA response element (TEE). Both the Jun and Fos proteins are phosphoproteins, and their level of phosphorylation is affected by various stimuli [ 14*-,151. Extracellular signals modulate AP-1 activity by several distinct mechanisms. AU of the jun and fi genes are immediate-early genes, the transcription of which is
rapidly induced in response to cell stimulation with TPA, growth factors, cytokines, and other agents [ 131. Only in the case of C-J?%and c-jun have the promoter elements involved in these induction responses been determined. Most agents that lead to c-& induction do so by affecting the activity of a protein known as the serum response factor (SRF) that binds to the serum response element (SRE). Activity of SF@ is believed to be regulated by a post-translational mechanism, as it exists in the cell prior to the initial stimulus [ 161, but the exact mechanism of [email protected]
remains to be determined. Other agents such as CAMP and Ca2+ appear to induce [email protected]
CREB which recognizes a CRE within the clfos promoter [8-l. In most cell types, induction of c-Ji transcription is very rapid and highly transient [ 171. The same applies for the c-Fos protein which can be detected after 1 h of the initial stimulus and then disappears rapidly with a very short half-life . Induction of c-jun on the other hand is usually longer lasting than that of cl/bs and dependent on the cell type and the inducer; it varies from several to many hours or even days [ 19-211. Synthesis of c-Jun protein is delayed in comparison to induction of c-jun mRNA, and in the case of TPA treatment, increased c-Jun synthesis is detected only 2 h after the initial stimulus [ 191. Induction of c-jun transcription is mediated by a TRE present within its promoter [ 191. Experiments carried out in F9 embryonal carcinoma cells, which do not express c-Jun, suggest that c-jun induction by TPA requires the presence of pre-existing c-Jun protein [ 22 1. In contrast to the positive autoregulation of c-jun, transcription expression of c-fa appears to be negatively autoregulated [ 231. The requirement for pre-existing c-Jun protein for c-ju?l induction suggests that the initial response to TPA is mediated by a post-translational mechanism. Unlike F9 cells, other differentiated cell types exhibit considerable basal levels of c-Jun synthesis [ 14**,19] and recent studies indicate that c-Jun activity is post-translationally regulated [ 14**,24**]. Therefore, c-Jun is a likely candidate for the mediator responsible for c-jun induction. In nonstimulated cells, c-Jun is phosphorylated on three closely spaced sites that abut its DNA-binding domain. Phosphorylation of all three sites inhibits DNA binding. In response to TPA treatment, c-Jun undergoes rapid dephosphotylation on one or more of these sites, resulting in a protein that is more active in DNA binding [ 14**]. Several experiments strongly suggest that the kinase responsible for phosphocylation of these sites is glycogen synthase kinase (GSK)-3 and indeed, purified GSK3 phosphorylates c-Jun in vitro only on these three sites [ 14**] (J Wood gett et al., personal communication). Because GSK3 is predominantly a cytoplasmic enzyme, it appears that it phosphorylates c-Jun immediately after its synthesis and prior to its nuclear transfer. On the other hand, the TPAinduced dephosphorylation of c-Jun is likely to be mediated by a PKC-activated nuclear protein phosphatase [14**]. This enzyme, however, remains to be identified. In addition to phorbol esters, AP-1 activity is also induced in response to transient expression of various transforming oncogene products . These oncoproteins act at different steps along the same signaIIing pathway used
and gene control
by polypeptide growth factors . Furthermore, AP-1 activity is higher In transformed cells in comparison to their non-transformed counterparts . This elevation is mostly due to increased c-Jun expression and activity. In H-Ras transformed rodent fibroblasts, the rate of c-Jun synthesis is four-livefold higher than in their non-transformed parents. Synthesis of c-Fos on the other hand, is not affected. An even greater increase has been observed in c-Jun phosphorylation. Expression of activated H&s was found to have a complex effect on c-Jun phosphorylation. It results in both loss of phosphate from the inhibitory sites next to the DNA-binding domain, and increased phosphorylation of two other sites located within the c-Jun activation domain. The increased phosphorylation of the c-Jun activation domain correlates with its increased transcriptional stimulatory activity in response to H-Ras expression [24*-l. As H-FQs is known to activate at least two signal transduction pathways, only one of which involves PKC , it is not surprisir.g that it has a more complex effect on cJun phosphoryfation than TPA, a specific activator of PKC. Whereas TPA seems to affect only c-Jun DNA binding, H-Ras affects both the DNA-binding and transactivation functions of c-Jun [24**]. Because H-Ras mimicks and probably also mediates the action of growth factors , it is expected that agents such as plateletderived growth factor or epidennal growth factor will affect AP-1 activity by the same mechanism. These changes in the activity of pre-existing c-Jun protein should result in a cycle leading to further production of more c-Jun protein via the positive autoregulation of c-jttn transcription. This mechanism may account, at least in part, for the elevated levels of cJun synthesis in H-Ras transformed cells. Because primary rat embryo Iibroblasts are not eff~cienrly transformed by H-Ras alone, whereas the combination of H-Ras with deregulated c-Jun expression leads to efficient transformation [ 281, it appears that a yet to be discovered negative regulatory mechanism hinders the activation of c-Jun transcription by H-Ras in non-immortalized Iibroblasts. Such controls are probably lost in immortalized cell lines, such as NIH3T3, resulting in efficient transformation and activation of the endogenous cljun gene by H-Ras. c-Fos is also a phosphoprotein w-hose phosphorylation is stimulated by TPA [ 151. Although the TPA-inducible phosphotylation sites on c-Fos have not been mapped, most of them seem to be located at the carboxy-terminal region of c-Fos where its sequence diverges from that of the v-Fos protein. In support of this assignment, phosphorylation of v-Fos is not affected by TPA [ 151. The carboxy-terminal domain of the c-Fos protein is likely to contain its activation domain [ 291 and also appears to be involved in transrepression of the [email protected]
[ 231. Interestingly, conversion of several serine residues located within this region, which are likely to be phosphorylatlon sites, to alanines, prevents the ability of c-Fos to repress cfbs transcription without interfering with c-Fos mediated transactivation of a TRE-containing reporter [30**]. Thus, while phosphotylation of the c-Jun activation domain in-
and gene expression
creases its ability to stimulate transcription, phosphorylarion of the c-Fos activation domain seems to be required only for its transrepression activity. NFxB and IRF NFxB was originally thought to be a B-cell-specific transcription factor that binds to an enhancer element of the x light-chain immunoglobulin gene . Within a short time, however, it was found that NFxB activity can be induced in most cell types in response to activation of a variety of signal transduction pathways. Inducers of NFxB activity include phorbol esters, T-cell activators, cytokines, ultraviolet irradiation, and agents that activate the interferon (IPN) system, such as viruses and doublestranded RNA Many of the NFxB-inducible target genes encode cytokines and their receptors. It therefore appears that NPxB most probably has an important role in various intlammatory and immune responses. The active form of NPxB is a heterodimeric nuclear protein composed of 50 and 65 kD polypeptides [ 321. Both of these polypeptides participate in DNA binding. Interestingly, in non-stimulated cells, the majority of NPxB activity is present in the cytosolic fraction as a cryptic form which cannot bind DNA The cryptic form of NFxB can be activated by treatment of cytosolic extracts with deoxycholate or other agents that dissociate protein complexes . It was found that in the cytosolic fraction, the 50 and 65kD components of NFxB are associated with a 37 kD inhibitory protein, IxB. The association with IxB prevents the binding of the NPxB heterodimer to DNA and in vitro IxB can even dissociate a preformed NPxB-DNA complex [34-l. IxB appears to be a target for several protein kinases including PKA, PKC, and a heam-regulated kinase, all of which are capable of phosphorylating uncomplexed IxB in vitro and prevent its interaction with NFxB. IxB is accessible to these kinases even when associated with NFxB because incubation of the cryptic NFxB-IxB complex with any of these protein kinases in the presence of ATP leads to its dissociation [35,36**]. On the basis of these results, the following model was suggested to explain the activation of NFxB. In non-stimulated cells, the p50-p65 heterodimer is associated with the non-phosphorylated form of IxB in the cytosol. In response to extracellular signals that lead to activation of one of several protein kinases, including PKA and PKC, IxB undergoes phosphorylation which causes its dissociation from the p50-p65 heterodimer. The free NPxB heterodimer is then capable of translocation to the nucleus where it binds to speciIic sites in the regulatory regions of downstream genes, resulting in their induction. The mechanism responsible for the permanent activation of NPxB in B-cells remains to be elucidated, but it might be due to Inactivation of IxB or lack of its expression. While for the past several years NFxB was thought to be regulated exclusively by this post-translational mechanism, new lindings demonstrate the existence of additional mechanisms controlling NFXB activity. Recently, the ~50 cDNA was cloned and found to be a member of
the relgene family [ 37*,38*]. Interestingly, the open reading frame of this cDNA encodes a much larger protein of 105 kD. While the full-length protein does not bind to DNA, a truncated form similar to p50 is fully active in DNA binding. These Endings suggest that ~105 is the precursor for p50 and that its maturation requires a specitic proteolytic cleavage. Although it is not known whether this proteolytic reaction is subject to regulation, it is the first example of activation of a transcription factor by proteolysis. Using the p50 cDNA as a probe for RNA analysis, it was found that like other members of the rel family, which are known to be serum-inducible immediate-early genes 1391, the p50 gene is also inducible by the same stimuli known to activate NFxB [37*,40-l. The NFxB system is, therefore, analogous to the AP-1 system. Posttranslational events in both cases increase the ability of pre-existing protein to bind DNA and, in turn, this activated protein leads to synthesis of more protein by activating an autoregulatory loop. Analysis of the promoter of the p50 gene will determine whether it is a direct target for activation by NFxB. Positive autoregulation also seems to have a major role in controlling the activity of a third transcription factor, interferon regulatory factor (IRF)-1, responsible for the induction of interferon (IFN) genes in response to virus infection or double-stranded RNA . In addition to IRF-1, most cells express another protein, IRF-2, which has a primary structure quite similar to that of IRF-1. Although IRI-2 binds to the same response elements in IFN gene promoters, as does IRF- 1, unlike IRF-I it does not activate their transcription 1421. Induction of the IFN gene in response to virus infection and double-stranded RNA does not require de noL?oprotein synthesis and is thought to occur by a post-translational mechanism. Therefore, it seems quite surprising that expression of both IRF-1 and IRF-2 is induced in response to virus infection or treatment with IFN itself [41,42]. Recent studies shed new light on the mechanism of IPNgene induction and control of IRP-1 activity [43*]. Nonstimulated cells appear to contain low levels of inactive IRF-I and higher levels of active IRF-2. In response to virus infection, pre-existing IRF-I undergoes a post-translational modification that increases its DNA-binding activity, resulting in displacement of IRF-2 from the inactive IFN-gene promoters. The replacement of IRP-2 by IRP-1 leads to IFN-gene induction. This process seems to be facilitated by positive autoregulation of IRP-1 expression, which leads to production of more IRP-1 protein and results in more efficient displacement of IRP-2. Induction of IRP-1 gene expression, however, also occurs in response to various cytokines that are incapable of IPN gene induction. These lindings suggest that newly synthesized IRF-1 is deficient in either DNA binding or transcriptional activation and that full IRP-1 activity requires a post-translational modUication that is not part of the response to these cytokines but can be elicited by virus infection or double-stranded RNA which induces both IRP-1 and IPN transcription. A delayed induction of IRF-2 is supposed to be responsible for shutting off this induction response [43*]. This attractive model requires further substantiation by direct demonstration of IRF-1 activation by post-
translational modification. Identification of this modification reaction, and analysis of the IRF-1 promoter should clarify the signalling pathway involved in JFN gene induction. Interactions pathways
With the possible exception of IRF-1, the transcription factors described above are all involved in signal transduction pathways elicited in response to activation of cell surface receptors. Another important class of receptors involved in transcriptional regulation in response to extracellular signals are the nuclear receptors for steroid and thyroid hormones, vitamin D and retinoic acid. The signalling pathways are much less complicated than the ones used by ceU surface receptors because the nuclear receptors themselves are transcriptional regulators and the signals that activate them are transmitted by small am phiphilic molecules that can freely diffuse through lipid membranes and reach the nucleus . As indicated in all of the current textbooks on ceU biology the signalling pathways used by cell-surface and nuclear receptors are believed to be completely separate; however, five papers published almost simultaneously last September have totally revised this view and revealed the existence of an extensive crosstalk between these signalling pathways. While nuclear receptors are ligand-activated transcriptional activators responsible for the direct induction of many hormone-responsive genes by binding to hormone response elements (HREs) in their promoters, they are also potent inhibitors of the expression of other genes. One such gene is the human collagenase gene, the expression of which is induced by TPA, growth factors, and cytokines and is inhibited by glucocorticoids and retinoic acid. The cz3 element responsible for induction of collagenase gene transcription is the TRE [ 121. Interestingly, the same element is the target for negative regulation by retinoic acid  and glucocorticoids [ 46=,47**,48**,49*]. These findings suggested that the retinoic acid receptors and glucocorticoid receptor (GCR) are negative regulators of AP-1 activity. These predictions were verified by transfection experiments which demonstrated that the GCR can inhibit transcriptional activation by c-Jun and c-Fos. In addition, it was observed that both c-Jun and c-Fos irhibit gene activation by the GCR [46=,47**,48*=,49*). Because AP-1 does not bind to the HRE recognized but to the GCR, and GCR does not bind to the TRE [47**] it appears unlikely that this mutual inhibition is the result of competition between the two transcription factors for a common binding site. Two groups have actually been able to demonstrate that in Y&-Oc-Jun can inhibit DNA binding by the GCR, and that the GCR inhibits DNA binding by cjun or c-Jun plus c-Fos [47**,4&*). This inhibition most likely occurs by formation of an inactive protein complex involving the GCR, c-Jun and c-Fos. Evidence for formation of such a complex has been provided by crosslinking experiments [47**,50*].
and gene control
According to these lindings, the mutual antagonism at the gene level between the GCR and AP-1 does not require specialized response elements. The GCR will inhibit the binding of AP-1 to any TRE, and c-Jun or c-Fos will inhibit the binding of GCR to any glucocorticoid response element. Others, however, analysed another phorbol-ester and growth-factor regulated gene coding for proliferin, a putative growth factor. While previous work indicated that a classic AP-1 binding site that functions as the TRE of the prolifet-in gene is the target for negative regulation by glucocorticoids [ 511, a more recent study has identified a complex regulatory element upstream of this classic AP-1 binding site, as a second target for negative regulation [50.]. Multimers of this site form an inducible enhancer element that is activated by c-Jun and c-Fos and repressed by the GCR. This complex regulatory element seems to contain two low-affinity AP-1 binding sites that partially overlap a low-affinity GCR binding site. Because of its low al% ity, this element is not activated by the GCR alone, but is glucocorticoid-inducible when the GCR is expressed in the presence of cjun homodimers. When the GCR is expressed in the presence of c-Jun-c-Fos heterodimers, however, glucocorticoids repress the activation of this element by cjun-c-Fos. These complex effects led to the development of a second model to explain the antagonism between GCR and AP-1. According to this model, negative gene regulation by glucocorticoids requires a specialized response element to which both the GCR and AP-1 can bind simultaneously. When this element is occupied by GCR and c-Jun homodimers, it is glucocorticoidinducible, but when it is occupied by GCR and cgun-cFos heterodimers, its activity is repressed by glucocorticoids [50*]. Although each factor, however, has been shown to bind to this element by itself, simultaneous binding of GCR and AP-1 is yet to be demonstrated. This model also fails to explain the glucocorticoid repression of the simple TRE of the couagenase gene because the GCR does not bind to this site. Clearly, more work is required to determine the exact biochemical mechanism for the mutual antagonism between these two classes of transcription factors. Regardless of the exact biochemical explanation, however, the present findings illustrate the existence of an extensive crosstalk between the pathways used by polypeptide growth factors that act through ceU surface receptors and steroid hormones that act via nuclear receptors. Such interactions can account for the known ability of glucocorticoids to retard the growth of certain ceU types and prevent the activation of T cells. References Papers of have been . of .. of
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