Signalling within the
TRANSIENT SIGNALS generated by stimulation of cell-surface receptors are converted into long-term changes in gene expression by signal-regulated transcription factors that mediate the effects of polypeptide hormones, cytokines and neurotransmitters. The analysis of these 'nuclear messengers' has shed light on the final steps in cellular signal transduction and has thus offered a novel approach to tracing the critical events: start at the end and work backwards. Rapid advances in the study of transcription factors has given us great hope that, within a few years, most of the important steps in the transfer of information from the cell surface to the nucleus will be elucidated. Extracellular signals modulate the activity of many different types of transcription factors. One important group of signal-regulated transcription factors are the BZip proteins, so named because of their conserved basic (B) and leucine zipper (Zip) domains that are required for DNA binding and dimerization, respectivelyL These sequencespecific factors have a modular structure consisting of distinct and separable DNA binding, dimerization and transcriptional activation domains 2'3. The most studied members of this superfamily are the AP-1 (Jun/Fos) and CREB/ATF proteins that control gene expression by binding to the TPA (12-O-tetradecanoylphorbol-13-acetate) response element (TRE) and cyclic AMP (cAMP) response element (CRE), respectively3-z. Regulation by BZip proteins can involve a variety of complex mechanisms - transcriptional, combinatorial, temporal and post-translational that affect the level and the repertoire of the factors expressed in a given cell as well as their DNA binding and transcriptional activation functions. Because of the broad scope of this review we will focus on the mechanisms that modulate the activities of these factors in response to extracellular signals.
nucleus
TIBS 17 - OCTOBER 1992
Control of transcription factors by signal transduction pathways: the beginning of the end
Signal transduction pathways regulate gene expression by modulating the activity of nuclear transcription factors. The mechanisms that control the activity of two groups of sequence-specific transcription factors, the AP-1 and CREB/ATF proteins, are described. These factors serve as a paradigm explaining the transfer of regulatory information from the cell surface to the nucleus.
and responsible for transcriptional are initiated with the activation of induction of a number of genes in either tyrosine kinases or phospholipid response to activation of protein kinase turnover ~0. C (PKC) 3. Molecular cloning revealed The cAMP-regulated transcription that AP-1 consists of a collection of factor, CREB, was originally defined as a structurally related transcription factors, sequence-specific DNA activity that which belong to the Jun and Fos fami- binds to CREs within the promoters of lies; these associate to form a variety of cAMP-inducible genes and mediates homo- and heterodimers, all of which their induction in response to actirecognize the TRE3. Like all members of vation of the PKA pathway 5. Relatively the BZip family the AP-1 components soon it became clear that, in common must dimerize prior to DNA binding. with AP-1, CRE-binding activity is due to The Jun proteins bind DNA as either a number of closely related proteins n'~2. homodimers or Jun-Jun heterodimers, The family of CRE-binding proteins curwhereas the Fos proteins must hetero- rently consists of at least eight memdimerize with one of the Jun proteins, bers, of which only CREB has been since they cannot form stable Fos-Fos established as a mediator of cAMP homo- or heterodimers 3. Owing to their action 5. The exact function of other increased stability, the Jun-Fos dimers CRE-binding proteins, also known as exhibit more DNA-binding activity and ATFs, is not known. Also, AP-1 comtrans-activation capability than the cor- plexes can bind the CRE, albeit with responding Jun-Jun dimers 3,6. Among lower affinity in comparison to their the Jun proteins, c-Jun is the most po- interaction with the TREla. The CREB/ tent transcriptional activator, either as ATF proteins should be viewed as a a homodimer or in combination with c- large group of related transcription facFos 7'8. The Fos proteins also vary in their tors that bind to similiar target ability to activate transcription in com- sequences and, depending on the facbination with c-Jun, c-Fos and FosB being tor, can mediate the actions of cAMP, much more potent than Fral or Fra2 s,9. cellular and viral proteins like Ela, or Combinatorial interactions between the other unknown signals 12-14, Outside Jun and Fos proteins give rise to dimers their highly homologous DNA-binding with different activities, although their and dimerization domains, most of The compositionand functionof AP-1 and sequence specificity appears to be very these proteins have little, if any, homCREB/ATF AP-1 was originally defined as a DNA- similiar. All of these dimers are thought ology in their transcriptional activation binding activity recognizing the TRE to contribute to AP-1 activity and par- domains. Recently, a new member of this ticipate to varying extents in its regu- family has been isolated that is regulation by extracellular stimuli 3. AP-1 lated by tissue-specific and developM. Karin and T. Smeal are at the Department should be regarded as a nuclear mess- mentally controlled alternative splicing. of Pharmacologyand T. Smeal is also at the enger that mediates the actions of sig- Some splice variants of this factor, Department of Biology, Centerfor Molecular nal transduction pathways stimulated CREM, act as dominant-negative inGenetics, Universityof California, San Diego, School of Medicine, La Jolla, CA 92093-0636, by growth factors, hormones, cytokines hibitors of cAMP response elements, and neurotransmitters, most of which while others act positively ~5. USA. © 1992,ElsevierSciencePublishers. (UK) 0376-5067/92/$05.00 418
TIBS 17 - OCTOBER 1992 In general, the CRE-binding proteins do not bind TREs. However, several of them can form heterodimers with the Jun/Fos proteins that can bind both types of elements. For example, c-JunATF-2 (CRE-BP1) heterodimers bind to CREs with higher affinity than to TREs, while c-Jun-ATF-3 heterodimers fail to show this preference ~3. c-Jun therefore seems to be the most versatile, since it can heterodimerize with either ATF-2, ATF-3 or ATF-4, while c-Fos and Fral can only heterodimerize with ATF-4. This overlapping specificity of the AP-1 and CREB/ATF proteins is likely to play~a role in their ability to either antagonize or synergize with each other. Despite this overlapping binding specificity, for the most part CRE-dependent reporter genes respond to signals that activate the PKA pathway, whereas TRE-dependent reporters usually don't respond to such signals and instead are induced by stimuli that activate tyrosine kinases and/or PKC. Signals that regulate AP-1 and CREB/ATF
in addition to TPA, AP-] activity is induced by a variety of polypeptide hormones, growth factors, cytokines and neurotransmitters 3. These agents activate signalling pathways that are initiated with either stimulation of membrane-associated tyrosine kinases or phospholipid turnover, the latter giving rise to increased PKC activityTM. In addition, AP-1 activity is elevated in cells that express a variety of transforming oncogenes, whose products act as constitutively activated intermediates in the signal transduction pathway that transmits information from cell-surface tyrosine kinases to the nucleus. Such oncogene products include v-Src, HaRas, and v-Raf~6.Another class of agents that induce AP-1 activity share the common ability to induce oxidative stress (Ref. 3; Devary et al., submitted). These agents also seem to activate a signalling pathway that is initiated with the stimulation of membrane-associated tyrosine kinases (Devary et al., submitted). In all of these pathways, tyrosine-specific protein kinases act at the top, being either an integral part of cell-surface receptors or directly activated by interaction with occupied cell-surface receptors. The activation of tyrosine kinases results in a series of rather nebulous events that lead to increased Ras activity, which appears to play a pivotal role in the activation of downstream serine/threonine-specific protein kinases, such as Raf-1 and the ERKs~°,17J8(Fig. 1).
Signalling within the nucleus Cytokines
Growth factors (Sis)
-
?
'~~'--~
~
~
PTK
Raf-1 \
PKC
ERK-K ERK
/
p p / U l ~ p ~-kinase / J u n - kI i n a s e
/
CKII
' 63, 73
~
c
231,243, 249
cJun Figure 1
Regulationof c-Jun DNAbindingand transcriptionalactivity by extracellularsignals. In nonstimulated cells, c-Junis predominantlypresent in a latentform, which is mainlyphosphorylated on its inhibitorycarboxy-terminalsites, near its DNA-bindingdomain(DBD),while its amino-terminalactivation domain(hatchedbox) is hypophosphorylated.Diagramis greatly simplified, arrows (indicatingstimulation)do not neccessarilymean a single step nor are they exclusive. Numbersindicatethe residues in c-Junthat are subjectto phosphorylation. (Seetext for discussionof details.) These signalling pathways mainly affect AP-1 activity at two levels: transcriptional and post-translational. First, transcription of the fos genes, which is very low in most non-stimulated cells, is induced in response to a variety of extracellular stimuli. The most rapid induction is exhibited by c-fos, the expression of which is also highly transient while induction of other fos genes, such as fra-I is somewhat slower and longer lasting3,13. Most of the signals stimulate c-fos transcription through the serum response element (SRE) which is recognized by several different factors of which the major ones are p67SRF and p62TCF 19. It is still not clear how the activities of these constitutively expressed proteins are stimulated by extracellular signals. The promoters of other fos genes have not been analysed as thoroughly as the c-fos promoter. Induction of fos transcription results in increased syn-
thesis of Fos proteins, which combine with pre-existing Jun proteins to form more stable heterodimers and thereby increase the level of AP-1 binding activity3. Although most cells contain substantial levels of pre-existing Jun proteins that are responsible for their basal AP-1 activity, expression of some of the jun genes is also inducible 4,1°,29. Most of the signals that stimulate AP-1 activity induce c-jun transcription, which usually is longer lasting than c-fos induction3. The persistent induction of c-jun is presumbly due to the ability of c-Jun to autoregulate its expression by binding to a TRE in the c-jun promoter 2°. Expression of junB is also stimulated by extracellular stimuli but appears to respond to different signals than those which affect c-jun. For example, in 3T3 cells, c-jun transcription is stimulated by TPA and partially inhibited by cAMP, while junB (and c-fos) transcription is stimulated by
419
Signalling
w i t h i n the n u c l e u s
TIBS 17 - OCTOBER 1 9 9 2
gradually over 24 hours 4. This 'burst attenuation' kinetics closely parallels the phosphorylation state of CREB, which will be discussed in the following section. Also, ATF-1, which has a regulatory domain simfliar to the one of CREB, but lacks the glutamine activation domain present in CREB, is activated by the catalytic subunit of PKA22. The ATF-2 protein, which has a different regulatory domain, responds to a different type of signal: the Ela protein of adenovirus TM. Ela, which probably mimicks the activity of a (yet to be identified) physiological signal, binds to the activation domain of ATF-2 resulting in a large increase in its trans-activation potential.
Ligand
cAMP
Post-translational control by protein phosphorylation
/ [
(121), 133,
(,s6CREB
liiiiii!i! i !ii!i!iiiiii!iiii!iiiiiiiiiiiiiiiiiiiii!i!i c ili
Figure 2 The regulation of CREB activity in response to extracellular signals that elevate cAMP levels. The pathway that leads to the activation of PKA activity can be viewed as a simple twocomponent system as shown, cAMP binds to the regulatory subunit of PKA (R), allowing the catalytic subunit (C) to dissociate and translocate to the nucleus where it phosphorylates CREB on Ser133. The phosphorylation of Ser133 is attenuated by protein phosphatase 1, which is itself a substrate of PKA. The function of the other potential phosphorylation sites of CREB is unknown. Also, the calmodulin-dependent kinase has been shown to phosphorylate CREB in vitro, but the physiological significance of this event has not been established. Ser133 is located within a regulatory domain that affects the conformation and activity of the trans-activation domain (hatched box) located nearer the aminoterminus.
cAMP7. In most cells, expression ofjunD is constitutive 3, but in Hela cells its expression is induced only by the simultaneous activation of both PKC and PKA7. The differential responsiveness and induction kinetics of the various jun and fos genes results in the formation of different AP-1 complexes at different times after cell stimulation. The exact role of these dynamic changes in the composition and activity of AP-I is not fully understood, but at least part of them could be involved in termination of the induction response, as was shown for JunB, which can repress the activation of c-jun 7. In comparison to the AP-1 proteins, expression of the CREB/ATF proteins so far appears to be more or less constitutive and relatively unresponsive to extracellular stimuli. Therefore the major
420
level of control affecting these proteins is post-translational 4,5. As mentioned above, the only CREB/ATF protein with well-understood regulation is CREB itself. All of the signals known to stimulate CREB activity in vivo are those which activate the PKA pathway 4. The regulation of this pathway is much less complicated than that of signalling pathways centered around the Ras proteins (Fig. 2) 5. PKA activity is directly stimulated by elevation of cAMP levels; cAMP binds to the regulatory subunit of this tetrameric enzyme, leading to its dissociation and liberation of the catalytic subunit. This results in the activation of the catalytic subunit and its translocation to the nucleus where it can phosphorylate CREB21. The activation of CREB activity is rapid, generally peaking within 30 minutes and declining
CREB contains three phosphorylation sites in its amino-terminal half u,23, of which only the function of Ser133 is known. This residue is phosphorylated by PKA both in vitro and in vivo, resulting in a 10- to 20-fold increase in CREB's transcriptional activity23,24. Phosphorylation of this site by PICA follows the same kinetics as the induction of CREB target gene transcription 25. Phosphorylation of Ser133 does not detectably alter the DNA-binding affinity of CREB, and hybrid proteins containing CREB's amino-terminal half fused to the DNAbinding domain of GAL4 are activated by PKA. Phosphorylation of this site only affects, therefore, the activity of the trans-activation domain 23,26. Since substitution of Ser133 with acidic residues results in an inactive protein, phosphorylation is thought to activate CREB's trans-activation domain by altering its conformation, so that it can better interact with the transcriptional machinery 23. It is speculated that phophorylation occurs at a region of the protein that regulates the conformation of the trans-activation domain lying closer to the amino terminus (Fig. 2). The amino terminus of CREB, however, may be involved in cooperative interactions with other CREB dimers or some other DNAbinding protein, which would explain the finding that the increased expression of the tissue-specific extinguisher locus (TSE1), which encodes the regulatory subunit of PKA, not only reduces the phosphorylation of CREB at Ser133, but also inhibits occupancy of the CRE27. As yet, no function has been established for the other phosphoacceptor sites of CREB, Ser121 and Ser156. Although Ser121 and Ser133 can be
TIBS 17 - OCTOBER 1992
Signalling within
phosphorylated by PKC in vitro, there is no evidence that PKC phosphorylates CREB in vivo 24. Ser156 appears to be phosphorylated by casein kinase II (CKI1), but the functional relevance of this phosphorylation is unknown26. Although it was shown that Ser133 can be phosphorylated in vitro by the calmodulin-regulated CaM kinase28, there is no evidence that elevated intracellular calcium regulates CREB activity independently of the PKA pathway. Interestingly, transgenic mice that express a mutant CREB protein with Ala at position 133, exhibit a dwarf phenotype as a result of somatotrope hypoplasia29. Thus, phosphorylation of CREB at Ser133 appears to be required for proliferation of pituitary somatotropic cells. This is probably due in part to the induction of the homeobox gene GHFI by CREB3°, whose expression is required for proliferation of somatotropic cells3L Decreased GHF1 expression also leads to pituitary dwarfism32. These experiments provide strong physiological support for the role of Ser133 phosphorylation in mediating cAMP action in vivo. Although all of the AP-1 components are phosphoproteins, the role of phosphorylation in controlling their activity has been mostly worked out for c-Jun, which serves as a useful paradigm for its other family members, c-Jun is phosphorylated on at least five sites, two within its amino-terminal activation domain and three that are clustered next to its carboxy-terminal DNA-binding domain 33-38(Fig. 1). In a similiar manner to the phosphorylation of Ser133 on CREB, phosphorylation of Ser63 and Ser73 within the amino-terminal activation domain of c-Jun increases its ability to stimulate transcription 34-36. Phosphorylation rates of these sites are low in non-stimulated cells and are rapidly increased in response to growth factors such as PDGF or v-Sis, or expression of oncogenically activated Src, Ras and Raf proteins 34,35,38, In myeloid and lymphoid ceils, phosphorylation of these sites is stimulated by TPA36, but not in fibroblasts and epithelial cells 33. These differences may be due to different modes of Ha-Ras regulation in lymphoid cells versus fibroblasts 39. While phosphorylation of Ser133 in CREB results in a dramatic increase in its trans-activation function23, phosphorylation of Ser63 and Ser73 on c-Jun results in only a five- to tenfold increase in activity because the non-phosphorylated activation domain is already func-
tiona135,38. As shown for CREB, fusion of the activation domain of c-Jun to a heterologous DNA-binding domain generates a chimeric protein, the activity of which is stimulated by signals that enhance the phosphorylation of cJun 34'35'38. These signals enhance the trans-activation potential of the chimeric protein but do not affect its DNA-binding activity38. As shown for CREB, substitution of Ser63 and Ser73 with acidic residues does not result in a substantial increase in c-Jun activity (T. Smeal and M. Karin, unpublished). This suggests that phosphorylation of CREB and c-Jun potentiates trans-activation by affecting the conformation of the activation domain rather than by contributing additional negative charges that participate in electrostatic interactions with a component of the transcriptional machinery. One difference between CREB and c-Jun is that in the former the phosphorylation site lies outside the activation domain, while in the latter the phosphorylation sites are within the activation domain. However, in both cases, phosphorylation appears to act positively by increasing the ability of the activation domain to interact with a (yet to be identified) target that is part of the transcriptional machinery, rather than liberating them from the influence of a negative regulator. The kinase that phosphorylates the amino-terminal sites of c-Jun remains to be identified. Although one report suggests that these sites are phosphorylated by the 42kDa and 44kDa ERK (MAP2) kinases and a related 54kDa kinase36, several other groups find that the ERK (MAP2) kinases phosphorylate one of the carboxy-terminal inhibitory sites of c-Jun (see below) rather than the amino-terminal sites 4°,41. A recent report suggests that the kinase might be a 69 kDa protein, which is antigenically unrelated to the ERK proteins 42. Regardless of the identity of the kinase, it is agreed that its activity is regulated by the same signalling pathway used by cell surface tyrosine kinases and the Ras proteins. The carboxy-terminal phosphorylation sites of c-Jun were identified as Thr231, Ser243 and Ser24933,37. These sites are clustered amino-terminally to the basic region of c-Jun and this cluster contains a fourth phosphoacceptor site, Thr239, which does not appear to be phosphorylated in vivo 37. Two of the sites, Thr231 and Ser249, are phosphorylated by CKI137,whereas the third
the nucleus
site, Ser243, is phosphorylated, in vitro, by the ERK kinases4°.4L In vitro phosphorylation of Thr231 and Ser249 by CKII inhibits the DNA-binding activity of c-Jun in a reversible manner and injection of peptides that inhibit or titrate CKII activity induces the expression of a AP-1 dependent reporter 37. Neither the inhibitory peptides nor CKIIwere found to have an effect on the expression of a CREB-responsive reporter, thereby providing further indication that phosphorylation of CREB by CKIF6 may not be of regulatory importance. While the exact mechanism by which phosphorylation of the carboxy-terminal sites of c-Jun inhibits DNA binding remains to be identified, their close proximity to the basic region suggests that inhibition is due to electrostatic repulsion between phosphates on c-Jun and those of the DNA backbone.
The role of phosphatases In general, much less is known about the importance of protein phosphatases, in comparison to protein kinases, in regulating transcription. However, it can be easily predicted that phosphatases should, in general, counteract the effects of protein kinases. One problem, however, with this simple assumption is that the specificity of the protein phosphatases may not be as narrow as that of protein kinases. On the other hand, studies with both AP-1 and CREB indicate that phosphatases affect these proteins in a rather specific manner. The major kinase that phosphorylates the carboxy-terminal sites of c-Jun, CKII, is a constitutive protein kinase37, while other kinases that phosphorylate this region, the ERKs, exhibit inducible activity's. Cell stimulation with TPA or expression of activated Ha-Ras results, however, in rapid dephosphorylation of two of the carboxy-terminal sites 33-35. These results strongly suggest that these stimuli activate a phosphatase specific for the carboxy-terminal sites of e-Jun, rather than inhibit the kinases that phosphorylate this region 37. Although the Jun-phosphatase remains to be identified, it appears to have an important role in the regulation of AP-1 activity and c-]un expression. In nonstimulated HeLa cells, c-Jun is the predominant component of AP-1. In these cells, independently of new protein synthesis, c-jun transcription is rapidly activated by PKC stimulation, acting through the aforementioned TRE, in the c-jun promoter 2°. The same treatment results in a parallel increase in AP-1 bind421
Signalling within ing activity, which is also relatively independent of new protein synthesis3. Since both of these events occur with similiar kinetics to the TPA-induced dephosphorylation of c-Jun 33, it appears that the role of the phosphatase is to increase DNA-binding activity by relieving the inhibitory effect of phosphorylation by CKII. In support of this hypothesis, substitution of Ser243 of c-Jun by Phe - which severely decreases its ability to be phosphorylated by CKII both in vitro and in v i v o 37 - results in a large increase in its trans-activation potential33. Presumably, following its dephosphorylation by the phosphatase, c-Jun binds to the TRE of the c-jun promoter, leading to increased c-jun expression. In the case of CREB, protein phosphatase 1 (PP1) has been implicated in terminating CREB's activation by cAMP25. As noted above, the induction of transcription by cAMP closely parallels the phosphorylation state of CREB at Ser133, suggesting that dephosphorylation of this site is responsible for the termination of the transcriptional reponse to cAMP. Treatment of cells with okadaic acid, a protein phosphatase inhibitor, after induction with cAMP agonists prevented CREB dephosphorylation and resulted in a corresponding increase in the transcriptional response of a reporter gene25. Similar results were obtained with a chimeric activator containing the amino-terminal activation domain of CREB, suggesting that the effect of okadaic acid was due to its effect on CREB phosphorylation. Most importantly, in vitro PP1 efficiently dephosphorylated CREB, while increased PP1 expression in viva decreased CREB activity. Furthermore, microinjection of the naturally occurring specific PP1 inhibitor-1 @1) resulted in the induction of CRE activity and augmented the response to subthreshold levels of cAMP25. Interestingly, I-1 activity is stimulated by PKA phosphorylation43, suggesting the existence of a negative regulatory loop whose function is to terminate CREB activation (Fig. 2). It is possible that the stimulation of the cJun activation domain is terminated in a similiar manner, via a phosphatase that acts on the amino-terminal phosphorylation sites. However, in this case, it is even more likely that a phosphatase is responsible for terminating the activation of the amino-terminal kinase, which is likely to depend on phosphorylation by upstream kinases (Fig. 1).
422
TIBS 17 - OCTOBER 1992
the nucleus
Concludingremarks In most cell types, the interactions between the predominant pathways that modulate AP-1 and CREB activity are antagonistic. Under special circumstances, however, in specific cell types, one can expect synergistic interactions as well. Undoubtedly there is more to be learned about the precise mechanisms that control the various AP-1 and CREB/ATF proteins and how they are fine-tuned. The exact functions of the many components of these redundant systems will require extensive analysis of 'knock-outs' where different BZip genes have been inactivated by homologous recombination. Another area that deserves extensive efforts is the identification of physiological target genes regulated by the different factors and their myriad of combinations. Only after such target genes are identified can we fully understand the regulatory potential of these systems.
5537-5542 10 Cantley, L. C. et al. (1991) Cell 64, 281-302 11 Gonzalez, G. A. et al. (1989) Nature 337,
749-752 12 Hal, T., Liu, F., Coukos, J. and Green, M. R. (1989) Genes Dev. 3, 2083-2090 13 Hal, T. and Curran, T. (1991) Prec. Natl Acad. Sci. USA 88, 3720-3724 14 Liu, F. and Green, M. (1990) Cell 61,
1217-1224 15 Foulkes, N. S., Mellstrom, B., Benusiglio, E. and Sassone-Corsi, P. (1992) Nature 355, 80-84 16 Herrlich, P. and Ponta, H. (1989) Trends Genet.
5, 112-115 17 Kyriakis, J. M. et al. (1992) Nature 358,
417-421 18 Boulton, T. G. et al. (1991) Cell 65, 663-675 19 Hipskind, R. A. eta/. (1991) Nature 354,
531-534 20 Angel, P., Hattori, K., Smeal, T. and Karin, M. (1988) Cell 55, 875-885 21 Nigg, E. A., Hiltz, H., Eppenberger, H. M. and Dutly, F. (1985) EMBO J. 4, 2801-2806 22 Rehfuss, R. P., Walton, K. M., Loriaux, M. M. and Goodman, R. H. (1991) J. Biol. Chem. 266,
18431-18434 23 Gonzalez,G. A. and Montminy, M. R. (1989) Cell
59,675-680 24 Yamamoto, K. K., Gonzalez, G. A., Biggs, W. H. and Montminy, M. R. (1988) Nature 334,
494-498 25 Hagiwara, M. et al. (1992) Cell 70, 105-113
Acknowledgements We thank our colleagues for many suggestions and comments. We apologize to those whose work was not cited due to the limitation of space. Work in the authors' laboratories is supported by the N1H and T. S. was supported by a Pharmacological Sciences Training Grant from the NIH.
References 1 Vinson, C. R., Sigler, P. B. and Mcknight, S. L. (1989) Science 246,911-916 2 Lamb, P. and Mcknight, S. L. (1991) Trends Biochem. Sci. 16, 417-422 3 Angel, P. and Karin, M. (1991) Biochim. Biophys. Acta 1072, 129-157 4 Montminy, M. R., Gonzalez,G. A. and Yamamote, K. (1990) Trends Neurasci. 13, 184-188 5 Karin, M. (1992) FASEB J. 6, 2581-2590 6 Smeal, T., Angel, P., Meek, J. and Karin, M. (1989) Genes Dev. 3, 2091-2100 7 Chiu, R., Angel, P. and Karin, M. (1989) Cell 59, 979-986 8 Nakabeppu, Y. and Nathans, D. (1991) Ceil 64, 751-759 9 Suzuki, T. et al. (1991) Nucleic Acids Res. 19,
26 Lee, C. Q., Yun, Y., Hoeffler, J. P. and Habener, J. F. (1990) EMBO J. 9, 4455-4465 27 Boshart, M., Weih, F., Nichols, M. and Schutz, G. (1991) Ceil 66, 849-859 28 Sheng, M., Thompson, M. A. and Greenberg, M. E. (1991) Science 252, 1427-1430 29 Struthers, R. S. et al. (1991) Nature 350, 622--624 30 McCormick, A., Brady, H., Theill, L. E. and Karin, M. (1990) Nature 345,829-832 31 Castdllo, J., Theill, L. E. and Karin, M. (1991) Science 253, 197-199 32 Li, S. et al. (1990) Nature 347,528-532 33 Boyle, W. J. et al. (1991) Cell 64, 573-584 34 Binetruy, B., Smeal, T. and Karin, M. (1991) Nature 351,122-127 35 Smeal, T eta/. (1991) Nature 354, 494-496 36 Pulverer, B. J. eta/. (1991) Nature 352, 635-638 37 Lin, A. et al. Cell (in press) 38 Smeal, T. et al. (1992) Mol. Cell. Biol. 12, 3507-3513 39 Downward, J. et al. (1990) Nature 346, 719-723 40 Alvarez, E. et al. (1991) J. Biol. Chem. 266, 15277-15285 41 Ferrell, J. Mol. Biol. Cell (in press) 42 Adler, J., Franklin, C. C. and Kraft, A. S. (1992) Prec. Natl Acad. Sci. USA 89, 5341-5345 43 Cohen, P. (1989) Annu. Rev. Biochem. 58, 453-508
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TRANSIENT SIGNALS generated by stimulation of cell-surface receptors are converted into long-term changes in gene expression by signal-regulated transcription factors that mediate the effects of polypeptide hormones, cytokines and neurotransmitters. The analysis of these 'nuclear messengers' has shed light on the final steps in cellular signal transduction and has thus offered a novel approach to tracing the critical events: start at the end and work backwards. Rapid advances in the study of transcription factors has given us great hope that, within a few years, most of the important steps in the transfer of information from the cell surface to the nucleus will be elucidated. Extracellular signals modulate the activity of many different types of transcription factors. One important group of signal-regulated transcription factors are the BZip proteins, so named because of their conserved basic (B) and leucine zipper (Zip) domains that are required for DNA binding and dimerization, respectivelyL These sequencespecific factors have a modular structure consisting of distinct and separable DNA binding, dimerization and transcriptional activation domains 2'3. The most studied members of this superfamily are the AP-1 (Jun/Fos) and CREB/ATF proteins that control gene expression by binding to the TPA (12-O-tetradecanoylphorbol-13-acetate) response element (TRE) and cyclic AMP (cAMP) response element (CRE), respectively3-z. Regulation by BZip proteins can involve a variety of complex mechanisms - transcriptional, combinatorial, temporal and post-translational that affect the level and the repertoire of the factors expressed in a given cell as well as their DNA binding and transcriptional activation functions. Because of the broad scope of this review we will focus on the mechanisms that modulate the activities of these factors in response to extracellular signals.
nucleus
TIBS 17 - OCTOBER 1992
Control of transcription factors by signal transduction pathways: the beginning of the end
Signal transduction pathways regulate gene expression by modulating the activity of nuclear transcription factors. The mechanisms that control the activity of two groups of sequence-specific transcription factors, the AP-1 and CREB/ATF proteins, are described. These factors serve as a paradigm explaining the transfer of regulatory information from the cell surface to the nucleus.
and responsible for transcriptional are initiated with the activation of induction of a number of genes in either tyrosine kinases or phospholipid response to activation of protein kinase turnover ~0. C (PKC) 3. Molecular cloning revealed The cAMP-regulated transcription that AP-1 consists of a collection of factor, CREB, was originally defined as a structurally related transcription factors, sequence-specific DNA activity that which belong to the Jun and Fos fami- binds to CREs within the promoters of lies; these associate to form a variety of cAMP-inducible genes and mediates homo- and heterodimers, all of which their induction in response to actirecognize the TRE3. Like all members of vation of the PKA pathway 5. Relatively the BZip family the AP-1 components soon it became clear that, in common must dimerize prior to DNA binding. with AP-1, CRE-binding activity is due to The Jun proteins bind DNA as either a number of closely related proteins n'~2. homodimers or Jun-Jun heterodimers, The family of CRE-binding proteins curwhereas the Fos proteins must hetero- rently consists of at least eight memdimerize with one of the Jun proteins, bers, of which only CREB has been since they cannot form stable Fos-Fos established as a mediator of cAMP homo- or heterodimers 3. Owing to their action 5. The exact function of other increased stability, the Jun-Fos dimers CRE-binding proteins, also known as exhibit more DNA-binding activity and ATFs, is not known. Also, AP-1 comtrans-activation capability than the cor- plexes can bind the CRE, albeit with responding Jun-Jun dimers 3,6. Among lower affinity in comparison to their the Jun proteins, c-Jun is the most po- interaction with the TREla. The CREB/ tent transcriptional activator, either as ATF proteins should be viewed as a a homodimer or in combination with c- large group of related transcription facFos 7'8. The Fos proteins also vary in their tors that bind to similiar target ability to activate transcription in com- sequences and, depending on the facbination with c-Jun, c-Fos and FosB being tor, can mediate the actions of cAMP, much more potent than Fral or Fra2 s,9. cellular and viral proteins like Ela, or Combinatorial interactions between the other unknown signals 12-14, Outside Jun and Fos proteins give rise to dimers their highly homologous DNA-binding with different activities, although their and dimerization domains, most of The compositionand functionof AP-1 and sequence specificity appears to be very these proteins have little, if any, homCREB/ATF AP-1 was originally defined as a DNA- similiar. All of these dimers are thought ology in their transcriptional activation binding activity recognizing the TRE to contribute to AP-1 activity and par- domains. Recently, a new member of this ticipate to varying extents in its regu- family has been isolated that is regulation by extracellular stimuli 3. AP-1 lated by tissue-specific and developM. Karin and T. Smeal are at the Department should be regarded as a nuclear mess- mentally controlled alternative splicing. of Pharmacologyand T. Smeal is also at the enger that mediates the actions of sig- Some splice variants of this factor, Department of Biology, Centerfor Molecular nal transduction pathways stimulated CREM, act as dominant-negative inGenetics, Universityof California, San Diego, School of Medicine, La Jolla, CA 92093-0636, by growth factors, hormones, cytokines hibitors of cAMP response elements, and neurotransmitters, most of which while others act positively ~5. USA. © 1992,ElsevierSciencePublishers. (UK) 0376-5067/92/$05.00 418
TIBS 17 - OCTOBER 1992 In general, the CRE-binding proteins do not bind TREs. However, several of them can form heterodimers with the Jun/Fos proteins that can bind both types of elements. For example, c-JunATF-2 (CRE-BP1) heterodimers bind to CREs with higher affinity than to TREs, while c-Jun-ATF-3 heterodimers fail to show this preference ~3. c-Jun therefore seems to be the most versatile, since it can heterodimerize with either ATF-2, ATF-3 or ATF-4, while c-Fos and Fral can only heterodimerize with ATF-4. This overlapping specificity of the AP-1 and CREB/ATF proteins is likely to play~a role in their ability to either antagonize or synergize with each other. Despite this overlapping binding specificity, for the most part CRE-dependent reporter genes respond to signals that activate the PKA pathway, whereas TRE-dependent reporters usually don't respond to such signals and instead are induced by stimuli that activate tyrosine kinases and/or PKC. Signals that regulate AP-1 and CREB/ATF
in addition to TPA, AP-] activity is induced by a variety of polypeptide hormones, growth factors, cytokines and neurotransmitters 3. These agents activate signalling pathways that are initiated with either stimulation of membrane-associated tyrosine kinases or phospholipid turnover, the latter giving rise to increased PKC activityTM. In addition, AP-1 activity is elevated in cells that express a variety of transforming oncogenes, whose products act as constitutively activated intermediates in the signal transduction pathway that transmits information from cell-surface tyrosine kinases to the nucleus. Such oncogene products include v-Src, HaRas, and v-Raf~6.Another class of agents that induce AP-1 activity share the common ability to induce oxidative stress (Ref. 3; Devary et al., submitted). These agents also seem to activate a signalling pathway that is initiated with the stimulation of membrane-associated tyrosine kinases (Devary et al., submitted). In all of these pathways, tyrosine-specific protein kinases act at the top, being either an integral part of cell-surface receptors or directly activated by interaction with occupied cell-surface receptors. The activation of tyrosine kinases results in a series of rather nebulous events that lead to increased Ras activity, which appears to play a pivotal role in the activation of downstream serine/threonine-specific protein kinases, such as Raf-1 and the ERKs~°,17J8(Fig. 1).
Signalling within the nucleus Cytokines
Growth factors (Sis)
-
?
'~~'--~
~
~
PTK
Raf-1 \
PKC
ERK-K ERK
/
p p / U l ~ p ~-kinase / J u n - kI i n a s e
/
CKII
' 63, 73
~
c
231,243, 249
cJun Figure 1
Regulationof c-Jun DNAbindingand transcriptionalactivity by extracellularsignals. In nonstimulated cells, c-Junis predominantlypresent in a latentform, which is mainlyphosphorylated on its inhibitorycarboxy-terminalsites, near its DNA-bindingdomain(DBD),while its amino-terminalactivation domain(hatchedbox) is hypophosphorylated.Diagramis greatly simplified, arrows (indicatingstimulation)do not neccessarilymean a single step nor are they exclusive. Numbersindicatethe residues in c-Junthat are subjectto phosphorylation. (Seetext for discussionof details.) These signalling pathways mainly affect AP-1 activity at two levels: transcriptional and post-translational. First, transcription of the fos genes, which is very low in most non-stimulated cells, is induced in response to a variety of extracellular stimuli. The most rapid induction is exhibited by c-fos, the expression of which is also highly transient while induction of other fos genes, such as fra-I is somewhat slower and longer lasting3,13. Most of the signals stimulate c-fos transcription through the serum response element (SRE) which is recognized by several different factors of which the major ones are p67SRF and p62TCF 19. It is still not clear how the activities of these constitutively expressed proteins are stimulated by extracellular signals. The promoters of other fos genes have not been analysed as thoroughly as the c-fos promoter. Induction of fos transcription results in increased syn-
thesis of Fos proteins, which combine with pre-existing Jun proteins to form more stable heterodimers and thereby increase the level of AP-1 binding activity3. Although most cells contain substantial levels of pre-existing Jun proteins that are responsible for their basal AP-1 activity, expression of some of the jun genes is also inducible 4,1°,29. Most of the signals that stimulate AP-1 activity induce c-jun transcription, which usually is longer lasting than c-fos induction3. The persistent induction of c-jun is presumbly due to the ability of c-Jun to autoregulate its expression by binding to a TRE in the c-jun promoter 2°. Expression of junB is also stimulated by extracellular stimuli but appears to respond to different signals than those which affect c-jun. For example, in 3T3 cells, c-jun transcription is stimulated by TPA and partially inhibited by cAMP, while junB (and c-fos) transcription is stimulated by
419
Signalling
w i t h i n the n u c l e u s
TIBS 17 - OCTOBER 1 9 9 2
gradually over 24 hours 4. This 'burst attenuation' kinetics closely parallels the phosphorylation state of CREB, which will be discussed in the following section. Also, ATF-1, which has a regulatory domain simfliar to the one of CREB, but lacks the glutamine activation domain present in CREB, is activated by the catalytic subunit of PKA22. The ATF-2 protein, which has a different regulatory domain, responds to a different type of signal: the Ela protein of adenovirus TM. Ela, which probably mimicks the activity of a (yet to be identified) physiological signal, binds to the activation domain of ATF-2 resulting in a large increase in its trans-activation potential.
Ligand
cAMP
Post-translational control by protein phosphorylation
/ [
(121), 133,
(,s6CREB
liiiiii!i! i !ii!i!iiiiii!iiii!iiiiiiiiiiiiiiiiiiiii!i!i c ili
Figure 2 The regulation of CREB activity in response to extracellular signals that elevate cAMP levels. The pathway that leads to the activation of PKA activity can be viewed as a simple twocomponent system as shown, cAMP binds to the regulatory subunit of PKA (R), allowing the catalytic subunit (C) to dissociate and translocate to the nucleus where it phosphorylates CREB on Ser133. The phosphorylation of Ser133 is attenuated by protein phosphatase 1, which is itself a substrate of PKA. The function of the other potential phosphorylation sites of CREB is unknown. Also, the calmodulin-dependent kinase has been shown to phosphorylate CREB in vitro, but the physiological significance of this event has not been established. Ser133 is located within a regulatory domain that affects the conformation and activity of the trans-activation domain (hatched box) located nearer the aminoterminus.
cAMP7. In most cells, expression ofjunD is constitutive 3, but in Hela cells its expression is induced only by the simultaneous activation of both PKC and PKA7. The differential responsiveness and induction kinetics of the various jun and fos genes results in the formation of different AP-1 complexes at different times after cell stimulation. The exact role of these dynamic changes in the composition and activity of AP-I is not fully understood, but at least part of them could be involved in termination of the induction response, as was shown for JunB, which can repress the activation of c-jun 7. In comparison to the AP-1 proteins, expression of the CREB/ATF proteins so far appears to be more or less constitutive and relatively unresponsive to extracellular stimuli. Therefore the major
420
level of control affecting these proteins is post-translational 4,5. As mentioned above, the only CREB/ATF protein with well-understood regulation is CREB itself. All of the signals known to stimulate CREB activity in vivo are those which activate the PKA pathway 4. The regulation of this pathway is much less complicated than that of signalling pathways centered around the Ras proteins (Fig. 2) 5. PKA activity is directly stimulated by elevation of cAMP levels; cAMP binds to the regulatory subunit of this tetrameric enzyme, leading to its dissociation and liberation of the catalytic subunit. This results in the activation of the catalytic subunit and its translocation to the nucleus where it can phosphorylate CREB21. The activation of CREB activity is rapid, generally peaking within 30 minutes and declining
CREB contains three phosphorylation sites in its amino-terminal half u,23, of which only the function of Ser133 is known. This residue is phosphorylated by PKA both in vitro and in vivo, resulting in a 10- to 20-fold increase in CREB's transcriptional activity23,24. Phosphorylation of this site by PICA follows the same kinetics as the induction of CREB target gene transcription 25. Phosphorylation of Ser133 does not detectably alter the DNA-binding affinity of CREB, and hybrid proteins containing CREB's amino-terminal half fused to the DNAbinding domain of GAL4 are activated by PKA. Phosphorylation of this site only affects, therefore, the activity of the trans-activation domain 23,26. Since substitution of Ser133 with acidic residues results in an inactive protein, phosphorylation is thought to activate CREB's trans-activation domain by altering its conformation, so that it can better interact with the transcriptional machinery 23. It is speculated that phophorylation occurs at a region of the protein that regulates the conformation of the trans-activation domain lying closer to the amino terminus (Fig. 2). The amino terminus of CREB, however, may be involved in cooperative interactions with other CREB dimers or some other DNAbinding protein, which would explain the finding that the increased expression of the tissue-specific extinguisher locus (TSE1), which encodes the regulatory subunit of PKA, not only reduces the phosphorylation of CREB at Ser133, but also inhibits occupancy of the CRE27. As yet, no function has been established for the other phosphoacceptor sites of CREB, Ser121 and Ser156. Although Ser121 and Ser133 can be
TIBS 17 - OCTOBER 1992
Signalling within
phosphorylated by PKC in vitro, there is no evidence that PKC phosphorylates CREB in vivo 24. Ser156 appears to be phosphorylated by casein kinase II (CKI1), but the functional relevance of this phosphorylation is unknown26. Although it was shown that Ser133 can be phosphorylated in vitro by the calmodulin-regulated CaM kinase28, there is no evidence that elevated intracellular calcium regulates CREB activity independently of the PKA pathway. Interestingly, transgenic mice that express a mutant CREB protein with Ala at position 133, exhibit a dwarf phenotype as a result of somatotrope hypoplasia29. Thus, phosphorylation of CREB at Ser133 appears to be required for proliferation of pituitary somatotropic cells. This is probably due in part to the induction of the homeobox gene GHFI by CREB3°, whose expression is required for proliferation of somatotropic cells3L Decreased GHF1 expression also leads to pituitary dwarfism32. These experiments provide strong physiological support for the role of Ser133 phosphorylation in mediating cAMP action in vivo. Although all of the AP-1 components are phosphoproteins, the role of phosphorylation in controlling their activity has been mostly worked out for c-Jun, which serves as a useful paradigm for its other family members, c-Jun is phosphorylated on at least five sites, two within its amino-terminal activation domain and three that are clustered next to its carboxy-terminal DNA-binding domain 33-38(Fig. 1). In a similiar manner to the phosphorylation of Ser133 on CREB, phosphorylation of Ser63 and Ser73 within the amino-terminal activation domain of c-Jun increases its ability to stimulate transcription 34-36. Phosphorylation rates of these sites are low in non-stimulated cells and are rapidly increased in response to growth factors such as PDGF or v-Sis, or expression of oncogenically activated Src, Ras and Raf proteins 34,35,38, In myeloid and lymphoid ceils, phosphorylation of these sites is stimulated by TPA36, but not in fibroblasts and epithelial cells 33. These differences may be due to different modes of Ha-Ras regulation in lymphoid cells versus fibroblasts 39. While phosphorylation of Ser133 in CREB results in a dramatic increase in its trans-activation function23, phosphorylation of Ser63 and Ser73 on c-Jun results in only a five- to tenfold increase in activity because the non-phosphorylated activation domain is already func-
tiona135,38. As shown for CREB, fusion of the activation domain of c-Jun to a heterologous DNA-binding domain generates a chimeric protein, the activity of which is stimulated by signals that enhance the phosphorylation of cJun 34'35'38. These signals enhance the trans-activation potential of the chimeric protein but do not affect its DNA-binding activity38. As shown for CREB, substitution of Ser63 and Ser73 with acidic residues does not result in a substantial increase in c-Jun activity (T. Smeal and M. Karin, unpublished). This suggests that phosphorylation of CREB and c-Jun potentiates trans-activation by affecting the conformation of the activation domain rather than by contributing additional negative charges that participate in electrostatic interactions with a component of the transcriptional machinery. One difference between CREB and c-Jun is that in the former the phosphorylation site lies outside the activation domain, while in the latter the phosphorylation sites are within the activation domain. However, in both cases, phosphorylation appears to act positively by increasing the ability of the activation domain to interact with a (yet to be identified) target that is part of the transcriptional machinery, rather than liberating them from the influence of a negative regulator. The kinase that phosphorylates the amino-terminal sites of c-Jun remains to be identified. Although one report suggests that these sites are phosphorylated by the 42kDa and 44kDa ERK (MAP2) kinases and a related 54kDa kinase36, several other groups find that the ERK (MAP2) kinases phosphorylate one of the carboxy-terminal inhibitory sites of c-Jun (see below) rather than the amino-terminal sites 4°,41. A recent report suggests that the kinase might be a 69 kDa protein, which is antigenically unrelated to the ERK proteins 42. Regardless of the identity of the kinase, it is agreed that its activity is regulated by the same signalling pathway used by cell surface tyrosine kinases and the Ras proteins. The carboxy-terminal phosphorylation sites of c-Jun were identified as Thr231, Ser243 and Ser24933,37. These sites are clustered amino-terminally to the basic region of c-Jun and this cluster contains a fourth phosphoacceptor site, Thr239, which does not appear to be phosphorylated in vivo 37. Two of the sites, Thr231 and Ser249, are phosphorylated by CKI137,whereas the third
the nucleus
site, Ser243, is phosphorylated, in vitro, by the ERK kinases4°.4L In vitro phosphorylation of Thr231 and Ser249 by CKII inhibits the DNA-binding activity of c-Jun in a reversible manner and injection of peptides that inhibit or titrate CKII activity induces the expression of a AP-1 dependent reporter 37. Neither the inhibitory peptides nor CKIIwere found to have an effect on the expression of a CREB-responsive reporter, thereby providing further indication that phosphorylation of CREB by CKIF6 may not be of regulatory importance. While the exact mechanism by which phosphorylation of the carboxy-terminal sites of c-Jun inhibits DNA binding remains to be identified, their close proximity to the basic region suggests that inhibition is due to electrostatic repulsion between phosphates on c-Jun and those of the DNA backbone.
The role of phosphatases In general, much less is known about the importance of protein phosphatases, in comparison to protein kinases, in regulating transcription. However, it can be easily predicted that phosphatases should, in general, counteract the effects of protein kinases. One problem, however, with this simple assumption is that the specificity of the protein phosphatases may not be as narrow as that of protein kinases. On the other hand, studies with both AP-1 and CREB indicate that phosphatases affect these proteins in a rather specific manner. The major kinase that phosphorylates the carboxy-terminal sites of c-Jun, CKII, is a constitutive protein kinase37, while other kinases that phosphorylate this region, the ERKs, exhibit inducible activity's. Cell stimulation with TPA or expression of activated Ha-Ras results, however, in rapid dephosphorylation of two of the carboxy-terminal sites 33-35. These results strongly suggest that these stimuli activate a phosphatase specific for the carboxy-terminal sites of e-Jun, rather than inhibit the kinases that phosphorylate this region 37. Although the Jun-phosphatase remains to be identified, it appears to have an important role in the regulation of AP-1 activity and c-]un expression. In nonstimulated HeLa cells, c-Jun is the predominant component of AP-1. In these cells, independently of new protein synthesis, c-jun transcription is rapidly activated by PKC stimulation, acting through the aforementioned TRE, in the c-jun promoter 2°. The same treatment results in a parallel increase in AP-1 bind421
Signalling within ing activity, which is also relatively independent of new protein synthesis3. Since both of these events occur with similiar kinetics to the TPA-induced dephosphorylation of c-Jun 33, it appears that the role of the phosphatase is to increase DNA-binding activity by relieving the inhibitory effect of phosphorylation by CKII. In support of this hypothesis, substitution of Ser243 of c-Jun by Phe - which severely decreases its ability to be phosphorylated by CKII both in vitro and in v i v o 37 - results in a large increase in its trans-activation potential33. Presumably, following its dephosphorylation by the phosphatase, c-Jun binds to the TRE of the c-jun promoter, leading to increased c-jun expression. In the case of CREB, protein phosphatase 1 (PP1) has been implicated in terminating CREB's activation by cAMP25. As noted above, the induction of transcription by cAMP closely parallels the phosphorylation state of CREB at Ser133, suggesting that dephosphorylation of this site is responsible for the termination of the transcriptional reponse to cAMP. Treatment of cells with okadaic acid, a protein phosphatase inhibitor, after induction with cAMP agonists prevented CREB dephosphorylation and resulted in a corresponding increase in the transcriptional response of a reporter gene25. Similar results were obtained with a chimeric activator containing the amino-terminal activation domain of CREB, suggesting that the effect of okadaic acid was due to its effect on CREB phosphorylation. Most importantly, in vitro PP1 efficiently dephosphorylated CREB, while increased PP1 expression in viva decreased CREB activity. Furthermore, microinjection of the naturally occurring specific PP1 inhibitor-1 @1) resulted in the induction of CRE activity and augmented the response to subthreshold levels of cAMP25. Interestingly, I-1 activity is stimulated by PKA phosphorylation43, suggesting the existence of a negative regulatory loop whose function is to terminate CREB activation (Fig. 2). It is possible that the stimulation of the cJun activation domain is terminated in a similiar manner, via a phosphatase that acts on the amino-terminal phosphorylation sites. However, in this case, it is even more likely that a phosphatase is responsible for terminating the activation of the amino-terminal kinase, which is likely to depend on phosphorylation by upstream kinases (Fig. 1).
422
TIBS 17 - OCTOBER 1992
the nucleus
Concludingremarks In most cell types, the interactions between the predominant pathways that modulate AP-1 and CREB activity are antagonistic. Under special circumstances, however, in specific cell types, one can expect synergistic interactions as well. Undoubtedly there is more to be learned about the precise mechanisms that control the various AP-1 and CREB/ATF proteins and how they are fine-tuned. The exact functions of the many components of these redundant systems will require extensive analysis of 'knock-outs' where different BZip genes have been inactivated by homologous recombination. Another area that deserves extensive efforts is the identification of physiological target genes regulated by the different factors and their myriad of combinations. Only after such target genes are identified can we fully understand the regulatory potential of these systems.
5537-5542 10 Cantley, L. C. et al. (1991) Cell 64, 281-302 11 Gonzalez, G. A. et al. (1989) Nature 337,
749-752 12 Hal, T., Liu, F., Coukos, J. and Green, M. R. (1989) Genes Dev. 3, 2083-2090 13 Hal, T. and Curran, T. (1991) Prec. Natl Acad. Sci. USA 88, 3720-3724 14 Liu, F. and Green, M. (1990) Cell 61,
1217-1224 15 Foulkes, N. S., Mellstrom, B., Benusiglio, E. and Sassone-Corsi, P. (1992) Nature 355, 80-84 16 Herrlich, P. and Ponta, H. (1989) Trends Genet.
5, 112-115 17 Kyriakis, J. M. et al. (1992) Nature 358,
417-421 18 Boulton, T. G. et al. (1991) Cell 65, 663-675 19 Hipskind, R. A. eta/. (1991) Nature 354,
531-534 20 Angel, P., Hattori, K., Smeal, T. and Karin, M. (1988) Cell 55, 875-885 21 Nigg, E. A., Hiltz, H., Eppenberger, H. M. and Dutly, F. (1985) EMBO J. 4, 2801-2806 22 Rehfuss, R. P., Walton, K. M., Loriaux, M. M. and Goodman, R. H. (1991) J. Biol. Chem. 266,
18431-18434 23 Gonzalez,G. A. and Montminy, M. R. (1989) Cell
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494-498 25 Hagiwara, M. et al. (1992) Cell 70, 105-113
Acknowledgements We thank our colleagues for many suggestions and comments. We apologize to those whose work was not cited due to the limitation of space. Work in the authors' laboratories is supported by the N1H and T. S. was supported by a Pharmacological Sciences Training Grant from the NIH.
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