Regulating transcription factor activity by phosphorylation

Regulating transcription factor activity by

phosphorylation

diverse effects on transcription factor activity and is responsible for bringing about changes in gene expression in response to a variety of environmental stimuli. The article is not an exhaustive survey of phosphorylation of all known transcription factors, but is intended to illustrate the main mechanisms by which protein phosphorylation can affect transcription factor activity.

Regulation of DNA binding by phosphorylation

The initiation of transcription by RNA polymerase II is controlled by transcription factors. Changes in gene transcription are brought about by regulating the activity of these factors. Phosphorylation of transcription factors as a regulatory mechanism is both rapid and readily reversible. Furthermore, because a transcription factor can be targeted by many protein kinases and phosphatases, phosphorylation can effectively integrate information carried by multiple signal transduction pathways, thus providing opportunities for great versatility and flexibility in gene regulation.

Stephen Jackson is at the Wellcome/CRC Institute of Cancer and Developmental Biology, Tennis Court Road, Cambridge CB2 I QR, UK.

The ability to regulate gene expression differentially in response to changes in the environment is of vital importance during cell growth and differentiation. In many instances, alterations in patterns of gene expression are triggered by agents such as growth factors interacting with receptors on the cell surface (Fig. 1). This stimulus is then transduced into the cytoplasm, where intracellular signalling systems are activated. The usual end point of such pathways is the activation or inactivation of a key molecule that regulates transcription 1, resulting in specific changes in gene transcription (Box 1). The observation that changes in gene expression can occur in the absence of protein synthesis indicates that they are often brought about by post-translational modification of transcription factors 3. Although transcription factors can be modified by reactions other than phosphorylation, such as glycosylation4, a number of lines of evidence indicate that phosphorylation is used most frequently to modulate their activity in response to environmental changes. First, many transcription factors are known to have their activity regulated by phosphorylation (see below). Second, signal transduction pathways that influence transcription factor activity frequently employ protein kinases (Fig. 1). Third, genetic approaches in organisms such as Saccharomyces cerevisiae have implicated both kinases and phosphatases in regulating transcription factor activity 5,6. In this review, I discuss recent evidence showing that phosphorylation has

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In many cases phosphorylation regulates the DNA-binding activity of a transcription factor. An example of phosphorylation negatively regulating DNA binding is provided by the proto-oncogene product c-Jun, which is a member of the AP-1 family of transcription factors. Since AP-1 activity is inducible by phorbol ester tumour promoters that activate protein kinase C (PKC), oJun is considered to be a final target for the PKC signal transduction pathway (Fig. 2). Phorbol ester induction of the DNA-binding activity of c-Jun appears to be achieved by specific dephosphorylation of three sites adjacent to the DNA-binding domain of c-Jun, rather than by direct phosphorylation of c-Jun by PKC 7. This is supported by the observation that glycogen synthase kinase 3 (GSK-3) phosphorylates the three sites in c-Jun in vitro and, in so doing, greatly diminishes the DNA-binding activity of c-Jun. Furthermore, mutation of one of the phosphorylated residues to a nonphosphorylatable phenylalanine residue results in loss of phosphorylation at all three sites and causes a large increase in c-Jun activity in vivo. It therefore appears that PKC activation by phorbol esters triggers a cascade of events that culminates in the removal of inhibitory phosphate moieties from c-Jun. This dephosphorylation might be achieved by inhibiting the activity of a GSK-3-1ike kinase. Alternatively, it might be mediated by the activation of a phosphatase that acts on cdun. Since the PKC pathway relays growth stimulatory signals within the cell, it is intriguing that the oncogenic derivative of Jun, v-Jun (of avian sarcoma virus 17), has a mutation in which one of the phosphorylation sites that affect DNA binding has been converted to a nonphosphorylatable phenylalanine. Perhaps this mutation uncouples v-Jun from normal cellular controls and thus contributes to its oncogenic potential. The regulation of DNA binding by AP-1 has recently taken on an additional layer of complexity with the discovery of IP-1, an activity that behaves as a trans-acting dominant inhibitor of DNA binding by AP-1 in vitro8. Interestingly, this factor functions as an inhibitor only when unphosphorylated, and it is inactivated by incubation with protein kinase A (PKA). These results therefore suggest that the DNA-binding activity of AP-1 can be regulated by both the PKA and PKC signal transduction pathways. This may enable integration of information conveyed by these two signalling systems. A factor whose DNA-binding activity is positively regulated by phosphorylation is the serum-response factor (SRF). SRF binds to the serum-response TRENDS IN CELL BIOLOGY VOL 2 APRIL 1992

element in the c-fos promoter and is thought to mediate the rapid increase of transcription of this 'immediate-early' gene in response to growth factors and mitogens. Indication that SRF is regulated by phosphorylation comes from the observations that phosphatase treatment reduces its DNA-binding activity, whereas phosphorylation in vitro by casein kinase II (CKII) increases its DNA-binding activity9, The site(s) phosphorylated by CKII has been mapped to a cluster of four serine residues that together regulate binding to DNA. Since the phosphorylated region is not within the minimal SRF DNA-binding domain and because phosphorylation appears to alter SRF protein conformation, an allosteric mechanism probably mediates the effect on activity 1°. Although CKII has not been unequivocally demonstrated to be the kinase acting on SRF in vivo, a number of lines of evidence support a model in which the DNA-binding activity of SRF in vivo is induced upon phosphorylation by CKII in response to serum stimulation 11. However, footprinting has shown that SRF is bound to DNA in vivo even before the serum response has occurred 12. This piece of apparently conflicting data has recently been explained by the observation that the effect of phosphorylation of SRF on DNA binding is not absolute: unphosphorylated SRF can bind to DNA but its binding and dissociation kinetics are much slower than those of its phosphorylated counterpart ]3. Indeed, at equilibrium, the difference in DNA-binding affinity between phosphorylated and unphosphorylated SRF is only two- to three-fold. In the light of these findings, it has been proposed that phosphorylation may allow inactive complexes present before stimulation by serum to be substituted by active ones containing phosphorylated SRF. Another example of phosphorylation having a quantitative rather than qualitative effect on binding to DNA is provided by the transcription factor encoded by the proto-oncogene c-myb. In this case, phosphorylation by CKII has no effect on the binding of c-Myb in vitro to high-affinity DNA sites that conform well to the optimal Myb-binding consensus sequence, but it inhibits binding to low-affinity sites 14. Phosphorylation of c-Myb may therefore allow the differential regulation of different subsets of Myb-responsive genes, regulating transcription of those containing weak binding sites, but not those containing strong binding sites. Thus, CKII has opposite effects on the DNA-binding ability of two different transcription factors: it increases SRF activity but decreases c-Myb activity. Finally, it is interesting to note that the c-Myb phosphorylation site is deleted in nearly all oncogenically activated Myb proteins, resulting in binding to DNA that is independent of CKII. Because CKII is modulated by growth factors, loss of this phosphorylation site might uncouple oncogenic Myb proteins from their normal physiological regulators. Despite much research, it is still unclear exactly how phosphorylation affects the binding of tranTRENDS IN CELL BIOLOGY VOL. 2 APRIL 1992

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Signal transduction pathways culminate in transcriptional regulation. Examples of intracellular signalling pathways that regulate transcription factor activity are shown. (left) The growth factor interacts with a transmembrane receptor and, in so doing, activates its cytoplasmic tyrosine kinase domain. The tyrosine phosphorylation of a cytosolic serine/threonine kinase then sets off a cascade of phosphorylation events that culminates in transcription factor phosphorylation. Components X and Y may be either cytoplasmic forms of transcription factors, or kinases that translocate into the nucleus. (right) The mitogen interacts with a cell surface receptor and mediates activation of adenylate cyclase via a G protein. The cAMP generated by this enzyme then activates PKA, which triggers events that culminate in transcription factor phosphorylation. These two pathways shown could act synergistically or antagonistically with one another by phosphorylating the same transcription factor, or they could affect the expression of different subsets of genes through phosphorylating different transcription factors.

scription factors to DNA. Since it affects some factors positively but others negatively, it is unlikely that the same mechanism operates in each case. For SRF, evidence suggests that phosphorylationinduced effects on binding to DNA are caused by changes in protein conformation. However, in some cases where there is negative regulation of binding it may be that the negative charge of the phosphate groups electrostatically hinders interaction with the similarly charged sugar-phosphate backbone of the DNA. 1 05

reviews BOX 1 - SEQUENCE-SPECIFIC REGULATORY TRANSCRIPTION FACTORS: MODE OF ACTION

by the transcription factor CREB (cAMP-responseelement-binding protein), which mediates transcription in response to activation of the cAMPdependent protein kinase (PKA) signal transduction pathway. PKA appears to activate the transcriptional activity (but not the DNA-binding activity) of CREB by phosphorylating CREB in-a domain required for transcriptional activationtS,16. A situation in vivo where this control operates is when forskolin treatment of rat PC12 cells increases cAMP production and thereby activates expression of the CREB-responsive somatostatin gene 15. In addition to responding to cAMP, it has recently been shown that CREB activity is also activated by phosphorylation in response to changes in intracellular calcium that occur upon membrane depolarization 17. In this case, phosphorylation of CREB is mediated by Ca2+-calmodulin-dependent protein kinases I and II. Interestingly, phosphorylation in response to calcium occurs on the same residue, Set 133, that is phosphorylated in other circumstances by PKA. Thus, information carried by two intracellular signalling pathways converges on a single residue / ~ /n s c / 'r i ( " p/ / " ~ ' ~/(~ and asstici~tf~c ~ within a transcription factor to induce the same effect. Another factor whose transcriptional activation properties are positively regulated by phosphorylation is c-Jun (Fig. 2). In this case, two sites located Initialion site Enhancer AP 1 Site GCBox TATA in the A1 transcriptional activation domain are Box phosphorylated in response to a variety of mitoI J i I gens and phorbol esters18,19. This seems to be Upstream Regulatory Factors General Transcriptional A p p a r a t u s mediated by the mitogen-activated protein kinases (MAP kinases), which respond to a variety of hormones and growth factors. In addition, phosR e g u l a t i o n o f t r a n s c r i p t i o n a l a c t i v a t i o n by phosphorylation of c-Jun on these sites is stimulated by phorylation oncogenic Ras proteins, suggesting that Ras might Phosphorylation can also influence transcription activate MAP kinases and thus activate c-Jun. This by regulating the ability of transcription factors to could explain the fact that Ras can cooperate with interact productively with other components of c-Jun in cell transformation and can activate tranthe transcriptional apparatus (Box 1). One of the scription through binding sites for AP-1. best examples of this type of regulation is provided There are a large number of other examples where phosphorylation has been implicated in regulating transcnpPKC pathway MAP kinase pathway tional activation by transcription factors but not their binding to DNA. For example, phosphorylation in vivo correlates positively with transcriptional competence for the GAL4 protein, the STE12 PP PP Dephosphorylation protein and the heat shock tranPhosphorylation of of DNA-binding domain transcriptional domain scription factor of S. cerevisiae20-22, b and for the human Oct-2 protein 23. DNA DNA In these cases, however, a causal relationship between phosphorylaDNA binding Activation Activation Activation + tion and increased activity has not yet been rigorously demonstrated. FIGURE2 One final example where phosRegulation of c-Jun activity by phospborylation. The DNA-binding activity of phorylation may be linked to tranc-Jun is negatively regulated by phosphorylation, whereas its transcriptional activation potential scriptional activation is provided by is positively regulated by phosphorylation. In response to growth stimulatory signals that the phosphorylation of Spl by the operate through the PKC pathway, three negative phosphate moieties on c-Jun are removed, DNA-dependent protein kinase 24. converting the protein into a form capable of binding DNA. To activate transcription, this form In this case, the kinase is itself a of c-Jun must be phosphorylated on two residues in its N-terminus. These latter DNA-binding protein and, furtherphosphorylations are catalysed by the MAP kinases. more, is active in vitro only when Analysis of transcriptional promoters for RNA polymerase II has revealed that the specificity (i.e. the precise selection of a start site) and efficiency of transcription is imparted by a variety of c/s-acting sequence elements that flank the transcriptional initiation siteI . One of these, the TATA box, binds the general transcription factor TFIID and directs the assembly of the general transcriptional apparatus on the promoter DNA2. Alone, this general transcription factor complex is capable of specific, albeit very low level, transcription. The efficiency of transcriptional initiation is affected by a group of sequence-specific DNAbinding regulatory transcription factors such as the Spl, Fos and Jun proteins indicated in the diagram. These factors can also stimulate transcription by binding to enhancer elements that are far away from the transcriptional initiation site (e.g. factor X in the diagram). Sequence-specific transcription factors are generally composed of two discrete protein domains: one binds DNA while the other influences transcriptional initiation via interactions with the general transcriptional apparatus (indicated by arrows). It is by regulating the activity of the upstream regulatory factors that transcription is modulated in response to physiological stimuli such as hormones, mitogens, morphogens and stress.

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TRENDS IN CELL BIOLOGYVOL. 2 APRIL 1992

bound to the same DNA molecule as Spl. Although the function of the DNA-dependent protein kinase in rive is not yet understood, it has been suggested that restricting phosphorylation to DNA-bound substrates might be a mechanism to activate factors such as Spl only when and where they are ready to engage in transcriptional activation. Phosphorylation may also affect the ability of transcription factors to serve as transcriptional repressers. An example of this is provided by another member of the AP-1 family, the proto-oncogene product c-Fos. In response to proliferative stimuli, c-fos transcription is rapidly activated, then quickly shut off again. This shut-off is mediated by a negative-feedback mechanism involving the c-Fos protein itself. It seems that only phosphorylated versions of c-Fos are able to inhibit transcription: mutated derivatives that are not efficiently phosphorylated can function as activators when dimerized to c-Jun, but cannot serve as autorepressors zs. Interestingly, c-Fos is approximately five-fold more phosphorylated than its viral oncogenic derivative v-Fos26, as v-Fos lacks the C-terminal phosphorylated region that is involved in autorepression. At present, it is not known in mechanistic detail how phosphorylation influences transcriptional activation potential. The observation that negatively charged amino acids are important for the activity of many transcriptional activation domains 1,27 initially suggested that phosphorylation may simply operate by providing negative charge. Although this may indeed sometimes be the case (for example, negatively charged amino acids can mimic phosphorylation of c-Fos2S), in other examples transcriptional effects appear to be mediated through alternative mechanisms is.

Regulation of subcellular localization by phosphorylation In addition to affecting transcription factor activity directly, phosphorylation may also influence the effective activity of transcription factors by regulating their subcellular localization. A wellcharacterized example of this is provided by the factor NF-~B. Although NF-~B was originally identified as a B-lymphocyte-specific factor responsible for activating immunoglobulin gene transcription, it was subsequently shown to be present in the cytoplasm of many other cell types in an inactive form that is unable to bind DNA 3 (Fig. 3). In this inactive state, NF-~cB exists in a stoichiometric complex with the inhibitory protein I~B. However, when cells are induced by agents such as the phorbol ester tumour promoters, which activate protein kinase C, NF-~cBbecomes able to enter the nucleus and activate specific gene transcription 28. Studies in vitro have shown that NF-~B activation can be achieved by dissociating the NF-~:B-hcB complex with detergents 29 or by phosphorylating I~B with PKC zs. This raises the possibility that PKC might directly affect NF-~:B relocation in vivo by dissociating its complex with I~B. Interestingly, phosphorylation by other kinases, such as the haemregulated eIF-2 kinase (HRI), can also lead to NF-~B TRENDS IN CELL BIOLOGY VOL. 2 APRIL 1992

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FIGURE3 Regulation of NF-~B localization by phosphorylation. NF-~cB exists in the cytoplasm of many nonlymphoid cells in an inactive complex with I~B that is excluded from the nucleus. Activation of a number of signal transduction pathways appears to result in phosphorylation of l~cBand dissociation

of the NI:-~B-I~Bcomplex. NF-•B is then able to enter the nucleus and activate transcription of specific genes. NF-KBis activated in vivo by phorbol esters (which act via the PKC pathway), interleukin 1 (which acts via the PKApathway) and double-stranded RNA[which activates a kinase with a specificity very similar to that of the elF-2 kinase (HRI)]. PKC and HRI,but not PKA,can directly phosphorylate and inactivate I~B in vitro. It has thus been suggested that PKA inactivates I~B through an intermediary component28. activation in vitro, suggesting that the NF-~cB-IKB system can integrate information from a number of signal transduction pathways z8 (Fig. 3). It has recently become clear that there is a family of NF-~B-related factors, some members of which appear to have their import into the nucleus regulated by I~B-related proteins 3°. Members of the NF-~cB family include the product of the protooncogene c-rel, and the Drosophila dorsal gene product. In the case of dorsal, control of nuclear localization plays an important role in establishing which regions of the developing Drosophila embryo become ventral structures 31. In this system, extracellular positional information in the developing organism is perceived by the Toll gene product, a cell surface receptor. Transduction of this stimulus is thought to result in phosphorylation of the cactus gene product (a member of the I~:B family), which then allows dorsal to enter the nucleus and activate transcription of a specific subset of genes. For the S. cerevisiae transcription factor SWIS, it is phosphorylation of the factor itself that affects subcellular localization. SWI5 is required for the cell-cycle-regulated transcription of the H e endonuclease gene that brings about yeast mating-type 1 07

reviews

interconversion. Although its activity is restricted to the G1 phase, SWI5 is present in yeast cells throughout the cell cycle. However, this factor is present in the yeast cell nucleus only during the G1 phase of the cell cycle, being confined to the cytoplasm in S, G2 and M phases 32. The regulated nuclear localization of SWI5 is imparted by a 50 amino acid region that acts as a Gl-phase-specific nuclear localization signal (NLS). SWI5 is phosphorylated in a cell-cycle-regulated manner on three sites within this region. It is phosphorylated when in the cytoplasm and dephosphorylated when in the nucleus 33. A likely kinase for SWI5 in vivo is the CDC28 protein, whose activity inversely correlates with the nuclear localization of SWI5 during the cell cycle, and which phosphorylates the three serine residues within the SWI5 NLS region in vitro. Consistent with these sites of phosphorylation directly affecting nuclear entry, their mutation to nonphosphorylatable residues converts SWI5 into a form that is constitutively nuclear in location 33. Although the precise mechanism is not yet understood, phosphorylation of SWI5 presumably renders its NLS unrecognizable by the nuclear transport machinery.

A versatile transcriptional control mechanism Phosphorylation has a number of features that make it ideal for regulating transcription factor activity. First, and perhaps foremost, phosphorylation can be exceedingly rapid: changes in c-Jun phosphorylation, for example, occur within 15 min of stimulating cells with phorbol esters 7. Second, it is readily reversible by phosphatases, permitting transcriptional regulation to operate in a highly dynamic fashion. Indeed, although this review has focused on kinases, it should be noted that phosphatases are not necessarily constitutively active, and that these may therefore also have important regulatory roles 34. Third, phosphorylation is very effective at integrating information from a number of signal transduction pathways. Fourth, in some cases a single kinase can have different effects on different transcription factors. Finally, depending on the amino acid residue of the target protein modified, phosphorylation can affect various aspects of transcription factor function. Given its versatility as a control mechanism, it is surely Acknowledgements inevitable that research will soon uncover many I thank more important examples where transcription facA. Bannister, tor activity is regulated by phosphorylation. C. Dingwall, R. Laskey, T. Kouzarides, D. St Johnston, R. Treisman and R. White for their helpful comments on this manuscript. Researchin my laboratory is funded by the Cancer Research Campaign. 1 08

References 1 MITCHELL,P. I. and TJIAN, R. (1989) Science245, 371-378 2 BURATOWSKI,S., HAHN, S., GUARENTE,L. and SHARP,P. A. (1989) Cell 56, 549-561 3 SEN,R. and BALTIMORE,D. (1986) Ce1147,921-928 4 JACKSON,S. P. and TJIAN, R. (1988) Ce1155, 125-133 5 HOEKSTRA,M. F., DEMAGGtO,A. J. and DHILLON, N. (1991) Trends Genet. 7, 256-260 6 ARNDT, K. T., STYLES,C. A. and FINK, G. R. (1989) Ce1156, 527-537 7 BOYLE,W. J. etaL (1991) Ce1164,573-584 8 AUWERX,J. and SASSONE-CORSI,P. (1991) Cell 64, 983-993

9 MANAK, J. R., DE BISSCHOP,N., KRIS,R. M. and PRYWES,R. (1990) Genes Dev. 4, 955-967 10 MANAK, J. R. and PRYWES,R. (1991) Mol. Cell Biol. 11, 3652-3659 11 GAUTHIER-ROUVIERE,C., BASSET,M., BLANCHARD,J-M., CAVADORE,J-C., FERNANDEZ,A. and LAMB, N. J. C. (1991) EMBOJ. 10, 2921-2930 12 HERRERA,R. E., SHAW, P. E. and NORDHEIM,A. (1989) Nature 340, 68-70 13 MARAIS,R. M., HSUAN,J. J., McGUIGAN, C., WYNNE, ]. and TREISMAN, R. (1992) EMBOJ. t 1, 97-105 14 LUSCHER,B., CHRISTENSON,E., LITCHFIELD,D. W., KREBS, E. G. and EISENMAN,R. N. (1990) Nature 344, 51 7-521 15 GONZALEZ, G.A. and MONTMINY, M. R. (1989) Cell59, 675-680 16 LEE,C. Q., YUN, Y., HOEFFLER,J. P. and HABENER,J. F. (1990) EMBOJ. 9, 4455-4465 17 SHENG,M., THOMPSON, M. A. and GREENBERG,M. E. (1991) Science252, 1427-1430 18 SMEAL,T., BINETRUY,B., MERCOLA,D. A., BIRRER,M. and KARIN, M. (1991) Nature 354, 494-496 19 PULVERER,B. J., KYRIAKIS,]. M., AVRUCH,J., NIKOLAKAKI,E. a~ld WOODGETT, J. R. (1991) Nature 353, 670-674 20 MYLtN, L. M., BHAT,J. P. and HOPPER,J. E. (1989) Genes Dev. 3, 1157-1165 21 SONG, O., DOLAN, J. W., YUAN, Y. O. and FIELDS,S. (1991) Genes Dev. 5, 741-750 22 SORGER,P. K. and PELHAM, H. R. B. (1988) Ce1154,855-864 23 TANAKA, M. and HERR,W. (1990) Cell60, 375-386 24 JACKSON,S. P., MacDONALD, J. I., LEES-MILLER,S. and TJIAN, R. (1990) Cell 63, 155-165 25 OFIR,R., DWARKI, V. ]., RASHID,D. and VERMA, I. M. (1990) Nature 348, 80-82 26 BARBER,J. R. and VERMA, I. M. (1987) MoL Cell. 8ioL 7, 2201-2211 27 PTASHNE,M. (1988) Nature 335, 683-689 28 GHOSH, S. and BALTIMORE,D. (1990) Nature 344, 678-682 29 BAEUERLE,P. and BALTIMORE,D. (1988) Ce1153,211-217 30 SCHMITZ, M. L., HENKEL,T. and BAEUERLE,P. (1991) Trends Cell BioL 1, 130-137 31 ST JOHNSTON, D. and NUSSLEIN-VOLHARD,C. (1992) Cell 68, 201-219 32 NASMYTH,K., ADOLF, G., LYDALL,D. and SEDDON,A. (1990) Cell 62, 631-647 33 MOLL,T., TEBB, G., SURANA,U., ROBITSCH,H. and NASMYTH, K. (1991) Ce1166, 743-758 34 TONKS, N. K. (1990) Curr. Opin. CellBiol. 2, 1114-1124

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