Cell, Vol. 80, 199-211, January 27, 1995, Copyright © 1995 by Cell Press Transcriptional Regulation Review by Extracellular Signals: Mechanisms and Specificity
Caroline S. Hill and Richard Treisman Nuclear Translocation Transcription Laboratory In principle, regulated nuclear localization of transcription Imperial Cancer Research Fund factors can involve regulated activity of either nuclear lo- Lincoln's Inn Fields calization signals (NLSs) or cytoplasmic retention signals, London WC2A 3PX although no well-characterized case of the latter has yet England been reported. N LS activity, which is generally dependent on short regions of basic amino acids, can be regulated either by masking mechanisms or by phosphorylations Changes in cell behavior induced by extracellular signal- within the NLS itself (Hunter and Karin, 1992). For exam- ing molecules such as growth factors and cytokines re- ple, association with an inhibitory subunit masks the NLS quire execution of a complex program of transcriptional of NF-KB and its relatives (Figure 1; for review see Beg events. While the route followed by the intracellular signal and Baldwin, 1993), while an intramolecular mechanism from the cell membrane to its transcription factor targets may mask NLS activity in the heat shock regulatory factor can be traced in an increasing number of cases, how the HSF2 (Sheldon and Kingston, 1993). When transcription specificity of the transcriptional response of the cell to factor localization is dependent on regulated NLS activity, different stimuli is determined is much less clear. How- linkage to a constitutively acting NLS may be sufficient to ever, it is possible to understand at least in principle how render nuclear localization independent of signaling (Beg different stimuli can activate the same signal pathway yet et al., 1992). activate different genes and how small differences in sig- nal strength can generate qualitative differences in gene DNA Binding expression. Many DNA-binding proteins bind DNA as oligomers, and In this review we shall concentrate on transcriptional signals can therefore regulate DNA binding by affecting responses to cell surface receptor-activated signaling factor oligomerization as well as protein-DNA interaction pathways; however, much of our discussion is also appli- itself. For example, signals may induce dissociation of tran- cable to signals induced by environmental stresses or to scription factors from inhibitory molecules that may either extracellular signals that act directly on transcription fac- directly block DNA-binding domain-DNA interactions, as tors, such as steroid hormones. Rather than describe in in the case of certain NF-~
SRF conserved Figure 1. Schematic Diagrams of Transcrip- Ets interaction SP/TP motifs tion Factors That Are Targets For Signaling TCF (Elk-l) Pathways
5133 Transcription factors are depicted by closed CREB bars with relevant domains indicated by stip- Q1 KID Q2 bZIP pling. Where the transcriptionfactor is a mem- S63/$73 T231JS243/S249 ber of a family, a single examplehas been il- lustrated. Hatching, DNA-binding domains; JUN --I ;, :::N\\'~,, 3~ 5 bz~ vertical lines, regulatedphosphorylation sites. SI-I3 S~ Y701 In Elk-l, the three stippled boxes indicatere- STAT 1 gions of homologybetween TCF family mem- bers. In CREB, the glutamine-richdomains that REL HOMOLOGY NLS are absentin CREMisoforms are indicated(Q1 NF-gB (p65) =~\\\\\\\\\\\\\\\\\'~,1 550 and Q2). The kinase-inducibledomain (KID) containsthe phosphorylatedSer-133 and mul- I~cBc~ tiple other phosphorylationsites. In Jun, the 8 MOTIFS ANKYRIN domain that is absent in v-Jun is indicated. In REL SIMILARITY STAT1 the DNA-bindingdomain has not been mapped. The regions of Src homology(SH2 and SH3) are indicated. In NF-KB the Rel homologydomain is shown, which is conserved in all other members of the family. The inhibitor of NF-~ potentiates transcriptional initiation remain only poorly un- this section we shall briefly review the representative derstood. For example, transcriptional activation by the examples of these strategies, which are summarized in extensively studied transcription factor CREB (cAMP re- Table 2. sponse element-binding protein) is dependent on regu- lated phosphorylation at Ser-133; this can be brought Activation in the Nucleus: MAP Kinase Pathways about independently by protein kinase A-, calmodulin-, or MAP kinases (MAPKs) are a family of protein.kinases nerve growth factor-induced kinases and is thus a conver- whose prototype members are the mammalian extracel- gence point for different signaling pathways (Gonzalez lular signal-regulated kinases ERK1 and ERK2 and the and Montminy, 1989; Sheng et al., 1991; Ginty et al., Saccharomyces cerevisiae pheromone-regulated kinases 1994). Ser-133 phosphorylation allows CREB to associate KSS1 and FUS3. MAPK phosphorylation sites contain the with a coactivator protein, CBP (Chrivia et al., 1993; Arias core sequence motif S/T-P. A number of nuclear transcrip- et al., 1994; Kwok et al., 1994); however, this does not tion factors have been identified as targets for MAPKs in appear to be sufficient for transcriptional activation, which metazoans, and MAPK signaling pathways provide rela- also involves neighboring glutamine-rich domains (Figure tively well-characterized examples of signaling cascades 1 ; for review see Lalli and Sassone-Corsi, 1994; see also with nuclear targets. Gonzalez and Montminy, 1989; Brindle et al., 1993; AI- The Ras/ERK MAPK Pathway berts et al., 1994a; Ferreri et al., 1994). In the case of Activation of receptor tyrosine kinases activates a signal- CREB, interaction with either specific cofactors like CBP ing cascade involving transient formation of ras-GTP and or the basal transcription machinery may be brought about activation of raf kinase at the membrane, followed by se- by a phosphorylation-induced conformational change in quential activation of MAPK kinase (MAPKK) and ERK1/ the protein (Gon zalez et al., 1991); phosphorylation-induced ERK2; only the latter enter the nucleus (for reviews see conformation changes may also be important in regulated Marshall, 1994; Leevers et al., 1994; Stokoe et al., 1994). activation by the TCF Elk-1 (see below; for references see In both mammalian cells and invertebrates, several tran- Treisman, 1994). scription factor targets of this pathway, which we shall refer to as the ras/ERK pathway, have been defined. In S. cerevisiae, a related pathway, coupled to a serpentine Transcription Factor Activation Can Occur in the receptor, activates the KSS1 and FUS3 MAPKs, causing Nucleus, at the Cell Membrane, activation of the STE12 transcription factor and transcrip- or in the Cytoplasm tion of pheromone-responsive genes (Herskowitz, 1995 [this issue of Cell]; see Table 3). Activation of transcription factors by extracellular signals In mammalian cells, the ras/ERK pathway provides a always involves a nuclear translocation step. Some path- common route by which signals from different growth fac- ways involve migration of signaling molecules themselves tor receptors converge at a major regulatory element of into the nucleus, while in others activated transcription the promoters of the c-foe and other coregulated genes, factors migrate to the nucleus following their activation in the serum response element (SRE). The major targets the cytoplasm. Moreover, although in many cases activa- for the pathway in the c-foe promoter are the TCF proteins tion of a single intracellular signaling pathway is sufficient Elk-1 or SAP-l, whose activity is regulated by phosphoryla- for transcription factor activation, some transcription fac- tion of a cluster of S/T-P motifs at their C-termini (see tors are complexes in which the activity of each component Table 1; Figure 1; for review see Treisman, 1994). SRE is regulated by different cellular signaling pathways. In mutations that prevent TCF binding substantially reduce 1~30 -N == (b "10 == Table 1. Properties of Transcription Factors That Are Targets for Signaling Pathways Transcription DNA-Binding DNA-Binding Properties and m Facter Family Domain Recognition Sites PropeFties Regulated by Signal Activating Signal x TCF (EIk-l,a Eta domain Binds SREs as ternary complex Transactivation; DNA binding Growth factors, via ras/ERK m SAP-1 ~) with SRF pathway c CREB/ATF bZIP Binds CREs as homodimer or as Transactivation; association with PKA pathway, growth factors (,9 (CREB ~) heterodimer with other ATF coactivator CBP 4 family members AP1 (Jun ") bZlP Binds TPA-responsive elements DNA binding; transactivation; UV irradiation, as homodimera or association with coactivator environmental stresses, heterodimers with other AP1 CBP? PKC activation family members STAT Novel Bind as homo- or heterodimers Dimerization; nuclear localization Cytokines, growth factors to GAS elements and related sites. STAT1 and STAT2 bind as part of heterotrimeric complex (ISGF3) to ISREs NF-KB Rel homology Binds KB sites as homo- or Nuclear localization; release from Lymphokines, TNFm PKC domain heterodimers with other family the inhibitor, IKB activation, members phytohemagglutinin, LPS, viral infection NF-AT complex; Rel-like NF-AT sites: composite element Nuclear localization (NF-ATp/c); T cell receptor activation contains (N F-ATp/c) containing NF-ATp/c and AP1 synthesis/t ransactivation (AP1) NF-ATp/c, AP1 bZIP (AP1) recognition motifs a Properties listed are for this family member. Cell 202 Table 2. Locationsof Transcription FactorsTargeted by ExtracellularSignals Transcription Location of Inactive Receptora Factor Target Signal Pathway TranscriptionFactor Growth factor TCF Ras/ERK pathway Nucleus Interferon, cytokine STAT JAK/STAT Cytoplasm, activatedat membrane TNFa NF-KB Unknown Cytoplasm T cell receptor NF-AT Calcium/PKC Cytoplasm (NF-ATp/c); (NF-ATp/clAP1) nucleus (AP1) aA specific exampleof a signalingpathway and transcription factor target is given in each case. c-fos promoter activation by growth factors and mitogens et al., 1994; Brunner et al., 1994; O'Neill et al., 1994). or by intracellular activators of the pathway such as v-raf In vitro, pointedP2 and yan are substrates for the ERK2 (Graham and Gilman, 1991; Kortenjann et al., 1994), but homolog ERKA/rolled (Brunner et al., 1994), while in trans- regulation can be restored to such mutants by expression fection experiments, activation of the MAPK pathway stim- of appropriately altered DNA binding specificity deriva- ulates activation by pointedP2 and inhibits a repressive tives of Elk-1 (Hill et al., 1994; Kortenjann et al., 1994). function of yan, suggesting that yan and pointedP2 may Biochemical studies indicate that Elk-1 is a good substrate have opposing effects on the same target promoters for ERKI/ERK2 in vitro, and the kinetics of its modification (Brunner et al., 1994; O'Neill et al., 1994). The genetic correlate well with MAPK activation in vivo (for review see data suggest that either loss of yan function or constitutive Treisman, 1994). Experiments with activated and interfer- dJun function is sufficient for R7 cell determination. One ing forms of MAPKK and ERK2 indicate that ERK activa- possible explanation for this is that both yan/pointedp2 and tion is necessary for activation of Elk-1 and SAP-1 in vivo dJun activate the same target promoters, perhaps through (Janknecht et al., 1993; Kortenjann et al., 1994). However, composite API/Ets motifs of the type first identified as a recent report indicates that SAP-1 may be activated by ras-responsive elements in some mammalian promoters ras/ERK-independent pathways in certain cells (Hipskind (Wasylyk et al., 1989; Gutman and Wasylyk, 1990). Reso- et al., 1994). Although phosphorylation can increase Elk-1 lution of this question awaits the identification of target DNA binding in vitro under certain conditions (Gille et al., promoters for the pointedP2, yan, and dJun proteins. 1992), the significance of this for SRE regulation remains The JNK/SAPK and Other MAPK Pathways unclear, since genomic footprinting indicates that an SRF- Studies of the mammalian Jun transcription factor led to TCF complex binds the c-fos SRE constitutively in vivo the identification of the JNK/SAPKs (for Jun N-terminal/ (Herrera et al., 1989). stress-activated protein kinases) as a family of MAPKs In Drosophila, the ras/ERK pathway apparently medi- distinct from the ERKs that are also regulated by extracel- ates cell fate determination in many different cell types. In lular signals (see Table 3; Hibi et al., 1993; D~rijard et al., the case of the Sevenless signaling pathway that controls 1994; Kyriakis et al., 1994; Davis, 1994). Transcriptional photoreceptor R7 cell fate, nuclear transcription factor tar- activation by Jun, as with SAP-1 and Elk-l, requires phos- gets of the pathway have been identified; these include phorylation at S/T-P motifs and can be potentiated by the the Ets domain proteins pointedP2 and yan and the Dro- Ras and Src oncoproteins (Figure 1 ; Binetruy et al., 1991 ; sophila Jun homolog dJun (for references see Bohmann Pulverer et al., 1991). However, the two S/T-P motifs that Table 3. Examplesof Transcription FactorsTargeted by MAPKs Stimulus MAPK Transcription Factor Target Mammaliancultured cellsa Growth factors, mitogens,cytokines ERKs(p42ERK1,p44ERK2) TCF UV light, stress, TNF(~, IL-1, osmotic shock JNK/SAPKs (p46, p54, p55) Jun UV light, stress, TNF(z, IL-1, osmoticshock, LPS p38/RK/MPK2. Unknown Growth factors p88FRKb FOS S. cerevisiae Mating pheromone KSS1, FUS3 STE12c Glycerol accumulation HOG1 Unknown Cellular integrity MPK1 Unknown Drosophilaphotoreceptor cells Boss/Sevenlessinteraction ERKNrolled PointedP2 (activator); yan (inhibitor); dJun (activator) a Not an exhaustivelist. In a particularcell, the different kinase subfamilies may be independentlyregulated. FRK is characterizedonly as a proline-directedkinase. ° It remainsunclear whether STE12 is a direct target for KSS1 or FUS3. Review: TranscriptionalRegulation by ExtracellularSignals 203 are phosphorylated in Jun are poor substrates for the ceptors during activation (Fu and Zhang, 1993; Lutticken ERKs but are efficiently phosphorylated by the JNKs/ et al., 1994). Phosphorylation at a conserved tyrosine at SAPKs (see below). The activity of the JNKs/SAPKs can their C-termini (see Figure 1) allows STAT dimerization; be regulated independently of that of the ERKs: agents an unknown mechanism then allows them to leave the that strongly stimulate Jun phosphorylation, such as ultra- receptor and migrate to the nucleus. violet light, strongly activate the J NKs/SAP Ks, but activate STAT activation by cytokine receptors lacking intrinsic the ERKs only weakly (Devary et al., 1992; Hibi et al., kinase activity involves specific members of the JAK (Ja- 1993; Radler et al., 1993; Kyriakis et al., 1994); moreover, nus kinase) family that are associated with the receptor dominant interfering ERK mutants do not inhibit Jun acti- (for review see Ihle et al., 1994; see Table 4). For example, vation (Westwick et al., 1994). The regulation of JNKs/ activation by interferons a and y (IFN(~ and IFNy) involves SAPKs varies among different cell types, since in T cells JAK1-TYK2-receptor or JAK1-JAK2-receptor complexes, JNK activation requires costimulation of both calcium- and respectively, and in both cases requires the presence of protein kinase C (PKC)-dependent pathways (Su et al., both kinases (Darnell et ai., 1994; Ihle et al., 1994). Al- 1994). As mentioned above, Jun DNA-binding activity is though many receptors activate the same JAKs, they acti- regulated by dephosphorylation of sites adjacent to its vate different STATs (see Table 4). For example, both the DNA-binding domain (Binetruy et al., 1991; Boyle et al., IFNa and interleukin-2 (IL-2) receptors activate JAK1, but 1991), but it remains unclear whether this event is con- STAT1 is activated only by the IFN(~ receptor (Beadling et trolled by the JNK/SAPK pathway. al., 1994). It is likely that specificity of interactions between In mammalian cells, at least two other proline-directed receptor phosphotyrosines and the STAT SH2 domain re- kinases appear to be regulated independently of the ERKs stricts which STATs can be recruited to a particular recep- and JNK/SAPKs: FRK, which phosphorylates the Fos tran- tor (Greenlund et al., 1994; Hou et al., 1994). The mecha- scriptional activation domain at Thr-232 (Deng and Karin, nism of STAT activation by receptor tyrosine kinases such 1994), and RK/MPK2/p38 (Freshney et al., 1994; Han et as the epidermal growth factor (EGF), ptatelet-derived al., 1994; Rouse et al., 1994), which appears to be the growth factor (PDGF), and colony-stimulating factor 1 (CSF1) vertebrate homolog of the yeast MAPK HOG1 (Table 3; receptors is less clear; however, EGF stimulation of A431 see Ammerer, 1994; Davis, 1994). Phosphorylation of cells does induce JAK1 phosphorylation (Shuai et al., CREB in response to nerve growth factor may also involve 1993), and STAT1 activation by EGF requires specific EGF a novel MAPK (Ginty et al., 1994). We shall address the receptor phosphotyrosines (Silvennoinen et al., 1993). question of how independently regulated pathways specif- Differential STAT activation leads to variation in tran- ically target similar substrates in a later section. scriptional activation owing to differential DNA target spec- ificity. The STAT proteins can bind DNA in two distinct Activation at the Membrane: JAK/STAT Pathways ways. First, different homo- and heterodimeric STAT com- The JAK/STAT pathways, which are involved in the tran- binations have subtly different sequence specificities and scriptional activation of many cytokine- and growth factor- bind sites related to the IFNy-activated sequence (GAS) inducible genes, illustrate how combinatorial interactions element (Ruff-Jamison et al., 1993; Sadowski et al., 1993; at the receptor itself can determine the kind of transcription Akira et al., 1994; Darnell et al., 1994; Wakao et al., 1994). complex activated and consequently the nature of the tar- Second, STAT proteins bind the ISRE (IFN-stimulated reg- get DNA sequence (for reviews see Darnell et al., 1994; ulatory element) as a heterotrimeric complex, ISGF3, Ihle et al., 1994). STAT proteins, which prior to receptor which contains STAT1 and STAT2 together with p48, a activation appear to be cytoplasmic (Schindler et al., member of the IRF-1 transcription factor family. Only the 1992), at least transiently associate with their cognate re- IFNa receptor, which can activate both STAT1 and STAT2, Table 4. Activation of JAKs and STATs by Different Cytokines and Growth Factors Receptor JAK Activated STAT Activated IFN~/J~ JAK1, TYK2 STAT1, STAT2, STAT3 IFNy JAK1, JAK2 STAT1 IL-3, GM-CSF, IL-5 (common I~ subunit) JAK2 Unknown IL-6, OSM, LIF, CNTF (common gp130 chain) JAK1, JAK2, TYK2 STAT1, STAT3 IL-2, IL-4, IL-7, IL-9 (common y subunit) JAK1, JAK3 IL-4 STAT/STAT6 IL-12 JAK2, TYK2 STAT4 EGF, PDGF, CSF1; receptor tyrosine kinases JAKla STAT1, STAT3b Epo JAK2 Unknown Growth hormone JAK2 Unknown Prolactin JAK1, JAK2 STAT5 G-CSF JAK1, JAK2 Unknown a JAK1 activation has been demonstrated only in the case of EGF, and its role in STAT activation remains unclear. b STAT3 activation has been demonstrated directly only in the case of EGF. Cell 204 can activate ISGF3 (Darnell et al., 1994). As yet, it is un- Many stimuli that activate NF-KB stimulate the production likely that the full repertory of STAT DNA-binding com- of reactive oxygen intermediates, while antioxidants sup- plexes and DNA targets has been established. press activation of NF-KB by a variety of stimuli; however, the mechanism by which reactive oxygen intermediates Activation in the Cytoplasm: NF-KB and Dorsal activate NF-KB remains obscure (for review see Baeuerle In mammalian cells activity of NF-KB is regulated by its and Henkel, 1994). association with an inhibitory subunit, IKB, which retains the inactive factor in the cytoplasm. NF-KB is activated Activation by Dual Signaling Pathways: NF-AT by many agents that induce acute phase responses, such A further level of complexity in signal-regulated transcrip- as IL-1, IL-2, tumor necrosis factor a (TNF~), bacterial tional activation occurs when activation of a single tran- lipopolysaccharide (LPS), viral infection, double-stranded scription factor requires signaling via more than one signal RNA, and activation of PKC; the factor is constitutively pathway, as in the case of NF-AT, the nuclear factor of active in B cells (for review see Baeuerle and Henkel, activated T cells, which regulates the promoters of cyto- 1994). In Drosophila, the recently identified NF-KB-related kine genes such as IL-2. Activation of NF-AT, which is a factor Dif also mediates immunity responses, while its bet- complex of two factors, requires activation of both calcium- ter characterized relative dorsal (dl) mediates dorsoventral and PKC-dependent signaling pathways by the T cell re- patterning (for references see St Johnston and Nesslein- ceptor (Flanagan et al., 1991; for review see Rao, 1994). Volhard, 1992; Ip et al., 1993). The NF-KB-type proteins One N F-AT component is a preexisting cytoplasmic factor, provide a case in which regulated factors either activate or NF-ATp/c, which is retained in the cytoplasm by an un- repress transcription in response to signals (for references known mechanism (McCaffrey et al., 1993; Northrop et see Beg and Baldwin, 1993; Jiang et al., 1993; Kirov et al., 1994). NF-ATp/c is activated by a calcium-dependent al., 1993; Lehming et al., 1994). pathway involving phosphatase PP2B/calcineurin: how- Genetic studies of Drosophila dl activation have pro- ever, although one of the candidate NF-ATp/c proteins is vided a framework for our understanding of NF-KB activa- a substrate for this enzyme (Jain et al., 1993), it remains tion. Translocation of dl to the nucleus is regulated by the to be shown that this dephosphorylation regulates factor interaction of the Toll receptor (whose cytoplasmic domain activity in vivo. The activation of NF-ATp/c appears to re- is related to that of the mammalian IL-1 receptor [Gay and quire solely its regulated nuclear transfer, since cyto- Keith, 1991 ]), with its ligand, Sp&tzle (Morisato and Ander- plasmic extracts from unstimulated cells are competent son, 1994). Like NF-KB, dl is retained in the cytoplasm to form the NF-AT complex when added to extracts con- by an hproteasome pathway (Beg et al., 1993; Cordle et al., induced is extensive, relatively little is known about how 1993; Henkel et al., 1993; Mellits et al., 1993; Miyamoto it is attenuated. Negatively acting factors may be present et al., 1994; Palombella et al., 1994). prior to stimulus or be synthesized as part of the response Given the diverse stimuli that activate NF-KB, it is possi- to it. Indeed, inhibition of protein synthesis potentiates the ble that there exist several pathways rather than a com- strength and duration of many signal-induced transcrip- mon messenger system and that the convergence point tional responses (see Greenberg et al., 1986), although between them may be the phosphorylation of IKB itself. these data must be interpreted cautiously since some pro- Some recent findings support this view. In HeLa cells, tein synthesis inhibitors themselves activate signaling selective ablation of the dou ble-stranded RNA-dependent pathways (Mahadevan and Edwards, 1991 ; Kyriakis et al., protein kinase (PKR) inhibits activation of NF-~CREBs for target sites (Mol- NF-KB inducers inactivate h Table 5. Down-Regulationof Signaling Pathways Inducible Pathway Gene Function Mechanism PKA ICER CRE-binding protein Inhibits CRE activity NF-KB IKB0c Inhibitory subunit of NF-KB Sequesters NF-~B in cytoplasm Growth factors Fos Component of AP1 Unknown; blocks SRE activity Growth factors, stress MKPI/CL100 Dual specificity phosphatase Inactivates ERKs, RK/p38 Mating pheromone MSG1 Dual specificity phosphatase Inactivates FUS3 (Sassone-Corsi et al., 1988). Examples of indirectly acting A major determinant of signal specificity is the substrate inducible proteins include yeast and mammalian dual- specificity of the kinases involved, about which relatively specificity phosphatases that inactivate MAPK following little is known. For example, substrates for the different pheromone or stress induction (Alessi et al., 1993; Sun proline-directed MAPKs cannot easily be distinguished on et al., 1993; Doi et al., 1994; Marshall, 1995 [this issue of the basis of sequences in the immediate vicinity of the Ce//]). phosphorylation sites. Recent studies of J NK/SAPKs sug- As yet little is known about the identity or regulation gest that additional physical interactions between the ki- of enzymes that inactivate transcription factors: many of nase and regions of the substrate distinct from the sites these will be phosphatases, although in the case of yeast of phosphorylation can also be important for specific sub- heat shock factor, specific induced phosphorylations may strate recognition. Deletion of the Jun 8 domain, a region mediate down-regulation (Hoj and Jakobsen, 1994). It is N-terminal to the phosphorylation sites, reduces the likely that phosphatase PP1 is responsible for the dephos- strength of interaction between Jun and JNK/SAPK in vitro phorylation of CREB Ser-133 (Hagiwara et al., 1992; AI- and abolishes N-terminal Jun phosphorylation in vitro and berts et al., 1994b) while phosphatase PP2A may be re- in vivo (Adler et al., 1992; Hibi et al., 1993). Different JNK/ sponsible for dephosphorylation of the TCF Elk-1 (for SAPK isoforms encoded by differentially spliced mRNAs references see Treisman, 1994). Although it remains un- appear to have different affinities for Jun, suggesting that clear whether the activity of CREB or TCF phosphatases they may have subtly different substrate specificities (Kal- is regulated by extracellular signals, it is interesting to note lunki et al., 1994; Sluss et al., 1994). However, it is not yet that a peptide inhibitor of PP1 can be inactivated by protein clear whether Jun and JNK/SAPKs form stable physical kinase A phosphorylation in vitro (Beullens et al., 1993). complexes in vivo (Hibi et al., 1993; Derijard et al., 1994). Activation of a transcription factor through phosphoryla- tion at multiple sites by the same kinase might also in Maintenance of Signal Specificity principle provide a mechanism by which signal specificity is maintained or even enhanced. Conceptually, this idea In this section we consider how signal identity is main- is similar to the use of multiple low specificity DNA-binding tained between related pathways that may share common subunits to generate complexes with increased DNA bind- components and discuss the factors that govern which ing specificity. A requirement for multiple modifications transcription factors respond to a particular pathway. would amplify small differences in the efficiency of phos- Specificity of this kind is essentially determined by the phorylation by different enzymes, especially if a phospha- specificity of the interactions between successive mole- tase were simultaneously active or kinase activity tran- cules in a signaling pathway: when the receptor for the sient. This would increase specificity at the bottom of a signal is itself the transcription factor, as in the nuclear pathway by setting a threshold for factor activation and hormone receptors, the problem disappears. would also allow tolerance of signal-independent noise or Association of signaling components into relatively sta- low level "cross-talk" between pathways. The TCF Elk-1 ble complexes is likely to play an important role in main- provides a potential example of this since its activation taining signal specificity by physically restricting access of requires phosphorylation at seven different S/T-P motifs signaling molecules to potential substrates. As discussed by ERK2 (for review see Treisman, 1994). above, even though the same JAKs may be activated by Finally, the DNA binding specificities of transcription fac- different receptors, the spectrum of STATs activated by tors themselves provide an important way by which the a particular receptor is apparently governed by specific targets for a signal pathway can be varied. Many factors physical interactions among STATs, JAKs, and receptors. bind DNA as dimers or heterodimers with related proteins, Similarly, in the S. cerevisiae pheromone response MAPK which may differ in DNA binding specificity. For example, pathway, the STE5 protein apparently acts as a docking Jun will bind with Fos to AP1 target sites but with ATF2 platform for other components of the cascade, which may or CREB to CRE-like sequences (Benbrook and Jones, limit cross-talk between it and the other MAPK pathways 1994), and different STAT dimers have subtly different (Ammerer, 1994; Choi et al., 1994; Marcus et al., 1994), sequence specificities (Ruff-Jamison et al., 1993; Sadow- while FUS3, one of the MAPKs at the end of this pathway, ski et al., 1993). Factor interactions may also enhance can be coprecipitated with its presumptive targets, includ- binding specificity. For example the TCFs, whose DNA- ing STE12 (Elion et al., 1993). binding domains have an inherently broad binding speci- Cell 206 ficity, are targeted to a subset of Ets motifs th rough cooper- example, if a single promoter contains multiple signal- ative DNA binding with SRF (for review see Treisman, regulated elements required for its activation, stimuli that 1994). A more extreme example of this is the recruitment can simultaneously activate all of these elements will acti- of transcription factors to target promoters by protein-pro- vate transcription, while stimuli that efficiently activate only tein rather than protein-DNA interactions, which may me- a subset of them will not. Similarly, if such interactions diate recruitment of STE12 to a-specific promoters in S. occur between factors regulated by different receptors, cerevisiae (Yuan et al., 1993). integration of signals from more than one receptor can occur. Combinatorial interactions between transcription Specificity of the Transcriptional Response factors bound at a promoter may also qualitatively change transcription factor behavior, in extreme cases affecting Our discussion so far has been limited to pathways and whether a signal-regulated transcription factor activates mechanisms that govern the activation of particular tran- or represses transcription. In addition, if such interacting scription factors in response to a given stimulus. What factors are themselves synthesized in response to the ini- determines which genes are activated by these factors, tial stimulus, activity of a target promoter can be prolonged and why is it that many stimuli induce the same signal or curtailed. pathways yet activate different genes? Several relatively Activation of the IFNI~ promoter provides a good exam- well-characterized examples suggest how combinatorial ple of how the requirement for multiple signal inputs at a interactions of signaling pathways and transcription fac- promoter can generate specificity. This promoter, which tors introduce specificity into the transcriptional response is activated following viral infection, contains several posi- to extracellular signals. tively and negatively acting elements, including an NF-KB site, that apparently direct formation of a highly organized Cell-Type Specific Transcriptional Regulation protein-DNA complex (for review see Thanes et al., 1993). The differential response of different cell types to signals The intact IFNI3 promoter is insensitive to many agents depends crucially on a cell's developmental history, which that can activate NF-KB, such as LPS and phorbol esters, manifests itself in two ways. First, target elements for regu- even though these agents can efficiently activate model lated transcription factors may either be in an accessible promoters containing multiple copies of the NF-KB site, chromatin conformation or inaccessible in condensed presumably because they cannot simultaneously activate chromatin. Second, combinatorial interactions of regu- the other IFNI3 regulatory elements. A similar mechanism lated factors with cell-type specific transcription factors may underlie the differential response of the c-fos gene will allow differential regulation in different cell types. to IFN(~ and PDGF: although both agents efficiently induce The pheromone response in S. cerevisiae provides an binding of STAT factors to the c-fos promoter, it is likely example in which such interactions are understood in de- that only PDGF can efficiently activate other cooperating tail. The differential response of a or a cells to pheromone c-fos promoter elements that are required for efficient tran- does not reflect differences in the signaling pathways acti- scription, such as the SRE (Hannigan and Williams, 1992). vated by the two cell type-specific pheromone receptors, Combinatorial interactions may allow genes to be in- because substitution of one receptor/pheromone combi- duced with different kinetics in response to the same stim- nation by the other has no effect on the spectrum of genes ulus. An example is the differential kinetics of transcrip- activated in a given cell (Bender and Sprague, 1986; see tional activation of the IL-2 and TNF(~ genes in response Herskowitz, 1995). In both a and c( cells, pheromone acti- to T cell receptor stimulation. Induction of IL-2 gene tran- vates the STE12 transcription factor via the MAPK path- scription is dependent on activation of both PKC- and cal- way, but some of the targets for STE12 are cell-type spe- cium-dependent signaling pathways and requires new cific, owing to the activity of the cell type-specific MAT protein synthesis. Both stimuli are required for activation proteins (see Herskowitz, 1995). In a cells, the MATal of the NF-AT complex and for full activation of AP1, which protein binds a-specific promoters, which allows STE12 is necessary for activation of other sites within the pro- recruitment; in contrast, a-specific promoters bind MAT(~2, moter; stimuli that activate the PKC-dependent pathway which prevents the access of STE12 to sequences within alone have no effect (for references see Lee and Gilman, these promoters by a system of chromatin-mediated re- 1994; Rao, 1994; Suet al., 1994). In contrast, activation pression involving the global transcriptional regulators of the TNFa promoter is more rapid, requiring only the SSN6 and TUP1 (see Cooper et al., 1994). In a cells, calcium-dependent pathway and the NF-AT component STE12 cannot be recruited to its (z-specific targets owing NF-ATp/c and occuring independently of new protein syn- to the absence of MATer1, but its a-specific targets are thesis (McCaffrey et al., 1994). In this case the differential now accessible owing to the lack of MATa2. timing of activation of the two genes in response to the same stimulus is determined by the different factor- Combinatorial Interactions Can Increase Specificity binding sites at the two promoters. Similarly, during growth of the Transcriptional Response factor stimulation of fibroblasts, synthesis of the egr-1 tran- The activity of many promoters apparently requires the scription factor prolongs the transcription of the subset of simultaneous presence of multiple transcription factors. other growth factor-induced genes that contain egr-1 sites Although the mechanistic basis of this requirement re- in their promoters, such as nur77(Williams and Lau, 1993). mains to be established, it is clearly a major determinant A good example of how simple combinatorial interac- of specificity in the response to extracellular signals. For tions can both modify transcription factor activity and inte- Review: Transcriptional Regulation by Ex~raceilular Signals 207 grate signal inputs from different signaling pathways is NLisslein-Volhard, 1992). In this case, differential activa- provided by the interaction of the Toll and torso pathways tion of the Toll receptor over the dorsoventral axis causes at Drosophila ventral repression elements (VREs) (Rusch the nuclear concentration of the dl morphogen to become and Levine, 1994). Although the Drosophila NF-KB homo- graded, being greatest in the ventral region and poles of log dl activates transcription in other promoter contexts, the embryo (St Johnston and N0sslein-Volhard, 1992; at VREs it acts with "corepressor" factors to inhibit tran- Rusch and Levine, 1994). At dl target promoters, the num- scriptional activation by other promoter elements. VRE ber and relative affinity of dl-binding sites, together with activity requires dl and is therefore dependent on the Toll cooperative interactions between dl and other transcrip- signaling pathway; it is also negatively regulated by the tion factors (which may act at the level of DNA binding torso receptor, acting via the MAPK pathway, which may or transcriptional activation), effectively sets a particular interfere with dl-corepressor interactions (Rusch and Le- threshold dl concentration for transcriptional activation vine, 1994). VRE-controlled genes such as zerknDllt (zen) (Figure 2; Jiang and Levine, 1993). This limits the expres- and decepentaplegic (dpp) are therefore repressed in ven- sion of key zygotic regulatory genes and thereby defines tral and lateral regions of the Drosophila syncytium where specific domains of the embryo. For example, promoters dl is activated by Toll, but remain active at the poles, where that contain only low affinity dl-binding sites are expressed the Toll and torso pathways are simultaneously active (see only where dl levels are highest, while those containing Figure 2, line 5; Jiang et al., 1993; Kirov et al., 1993; Lehm- high affinity dl sites together with other cooperating sites ing et al., 1994), are expressed even where dl levels are lowest (Figure 2, lines 1-3). Further specificity is provided by the combinato- Differential Effects of Signal Strength rial interaction of dl with a repressor, snail (sna), itself the or Duration: Thresholds product of a dl target gene. Promoters containing both dl Recent studies have demonstrated that PC12 cells can and sna sites, such as that of rhomboid (rho), are specifi- either differentiate or proliferate in response to growth fac- cally activated only in those regions of embryo in which the tor stimulation according to the strength or duration (or dl concentration is insufficient to induce sna expression both) of the stimulus (Traverse et al., 1994; Dikic et al., (Figure 2, line 4). Finally, as mentioned above, atthe poles 1994; Marshall, 1995). At present the mechanism of this the activity of dl as a repressor is regulated by a combinato- is unknown, but it is instructive to consider how small quan- rial interaction between the Toll and torso signaling path- titative differences in signal strength can be turned into ways (Figure 2, line 5). large qualitative differences in gene expression. Such phenomena might appear puzzling to signal transduction- Perspective ists, but come as no surprise to developmental biologists. The generation of dorsoventral polarity in the Drosophila The dl system provides the best example of how small embryo syncytium provides an example (St Johnston and differences in signal input can generate large differences ~derm [ neurectoderm ldorsal ectodemn Figure 2. Threshold Effects in Gene Activation by Drosophila DI HIGH Nuclear dl Threshold responses to the dl morphogen gra- concentration dient in the early Drosophila embryo. The curve represents the gradient of nuclear dl that is LOW , highest at the ventral midline (depicted by the vertical line). The three regions of tissue differ- PROMOTER TYPE entiation under dl control are indicated at the HIGH dl threshold top of the figure. Stippled and hatched bars 1. low affinity dl sites I indicate zones of dl-dependent activation and repression, respectively. The different kinds of MEDILI1VIdl threshold promoters that govern these zones of gene reg- high a f flnity dl sites (~-~oi) ulation are described at the left. (1) Activation 2. low affinity dl sites + E boxes (sna repzessor) of promoters containing only low affinity dl sites requires a high concentration of nuclear dl and LOW dl threshold therefore occurs only in the ventralmost region. 3. high affinity dl sites + E boxes (2) Activation of promoters such as those of twist(twl) or sna that contain either high affinity 4. high affinity dI sites + sna repressor sites (rho) dl sites or low affinity dl sites with collaborating sna on: sna off: repressed active E boxes, respectively, requires a lower concen- 5. VREs: high affinity dl sites+ corepressor sites ( zen, dpp) ~'JJJ/JJJfJJfJJJ. tration of nuclear dl and therefore occurs repression antagonized by torso pathway at poles throughout a more expanded region. (3) Activa- tion of promoters that contain both high affinity sites and collaborating E boxes requires only a low level of nuclear dl and occurs throughout the ventral region. (4) Some promoters in this latter class such as those of rho also contain sna-binding sites and are therefore repressed in the region, shown by a broken line, in which dl levels are high enough to activate the sna repressor gene: activation therefore only occurs in regions where sna is absent. (5) DI acts to repress transcription via VREs that are composed of high affinity dl-binding sites with sites for corepressors. Activity of genes containing VREs, such as dpp and zen, is repressed throughout the region where dl is nuclear; outside this region, the genes are activated by other promoter elements. At the poles, the torso pathway inactivates the VREs, and hence zen and dpp are expressed despite the high nucJear dl levels. Figure adapted from Jiang and Levine (1993). Cell 208 in transcriptional response. 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