Ternary complex factors: growth factor regulated transcriptional activators

Ternary complex factors: growth factor regulated transcriptional activators Richard Treisman Imperial

Cancer Research Fund, London,

UK

Members of a family of Ets domain proteins, the ternary complex factors (TCFs), are recruited to the c-fos serum response element by interaction with the serum response factor. Recent findings indicate that phosphorylation of TCFs occurs in response to activation of the MAP kinase pathway, and that regulation of TCF activity is an important mechanism by which the serum response element responds to growth factor signals. Current

Opinion

in Genetics

and Development

Introduction The serum response element (SRE) is a promoter element common to many cellular immediate-early gene promoters, and is activated both by growth factor stimulation and by oncogene products such as Src, Ras, Mos and Raf Ill. Recent studies have shown that many different growth factors activate cellular mitogen-activated protein kinases (MAP kinases) via a conserved kinase pathway under control of the Ras and Raf proteins, suggesting that the MAP kinase pathway is involved in SRE activation [see review by Marshall (pp 82-8911. At the c-fos SRE, a ternary complex forms between the ubiquitous transcription factor, serum response factor @RF), and a second protein, ternary complex factor (TCF) which cannot bind the SRE by itself 121. Mutational analysis of the C-$X SRE suggests that this complex is required for a full response to growth factor signals 12-41. The structure and properties of TCF are of considerable interest. In this review, I discuss recent data concerning the structure of TCF proteins, the mechanism of ternary complex formation, and the response of TCFs to growth factor stimulation. I then consider a number of outstanding questions concerning TCFs and the role of the ternary complex in signal transduction at the c-fos promoter.

Ternary

complex

factors

are Ets domain

proteins

TCF was initially identified by Nordheim and co-workers 121as a 62kDa polypeptide in HeLa cell extracts. Several cDNAs have been isolated that encode Ets domain proteins with DNA-binding properties identical to TCF, including SAP-l k51, Elk-l [6,71, SAP-2 (A Rogers, R Treisman, unpublished data) and NET (B Wasylyk, personal communication, also see 18.1).As well as their

MAP

kinase-mitogen-activated

96

response

.O Current

factor;

Biology

01

conserved amino-terminal Ets domains, these proteins contain two additional conserved regions, the B box and C box (Fig. la), and therefore constitute a distinct subclass of the Ets family (for a recent comprehensive review of Ets proteins, see 18.1). The Ets domain and the B box (a short hydrophilic region) mediate ternary complex formation and nuclear location [5,9-lO,ll**l. The proteins contain seven (S/T)P motifs (in the oneletter amino acid code), characteristic of sites that are phosphorylated by prolihe-directed kinases, five are located within the C box and two at the extreme carboxyl terminus, which is crucial for regulated transcriptional activation (112**-14**1; M Kortenjann, H Gille, P Shaw, personal communication). Figure lb shows the conserved carboxy-terminal sequences of Elk-l and SAP-l, including the (S/T)P motifs (note that the SAP-l sequence has been recently revised, SAP-la in Fig. lb; MA Price, R Treisman, unpublished data). Variants of SAP-l and Elk-l have been reported: in SAP-lb, the C box region is truncated, removing the six conserved carboxy-terminal (S/T)P motifs bl; while in AElk-1, amino acids 76-218 (including the carboxy-terminal part of the Ets domain and the B box) are deleted fl51.

DNA binding

and ternary

complex

formation

In the ternary complex at the human c-fos SRE, TCFs contact both SRF and an Ets motif, CAGGAT, 2 bp 5’ to the SRF site 12,5,7,161 (see Fig. 2). Elk-l and SAP-l can also bind DNA autonomously, but with restricted sequence specificity compared with that of the ternary complex; Ets motifs with an altered core sequence, CCGGAA, are high affinity autonomous binding sites [9,17*‘,181. Although a point mutation in the Elk-l Ets domain impairs autonomous binding in vitro, but not ternary complex formation, both abilities are abolished

Abbreviations protein kinase; RTK-receptor

SRF-serum

1994, 4:96-l

TCF-ternary

tyrosine kinase; SRE-serum complex

Ltd 0959-437X

factor.

response

element;

Ternarv

(a) Ets

domain structure

factors Treisman

protein Ternary complex formation *

Regulated transcription activation A

b

1 Interaction DNA

with

Interaction SRF

1

b with

Conserved P motifs

csm

Elk-l

1

90

148

168

352

399

428

aa

SAP-la

1

89

136

156

353

402

431

aa

carboxy-terminal

sequences

(b) Conserved Elk-l

353 363 368 383 389 I I SGSGLQAPG---P4LTPSLLPTHTLTPVLLTPSSLPPSSLPPSIHFWSTLSPIAPRSP~L-----SFQFPSjGSAQVHIPSISVDGLSTPWSPGPQKP :I 1 I::: I I::t SPLGILSPSLPTAljLTPAFF---SQTPIILTPSPLLSSIHFSTLSPVAPLSPARLQGANTLFQFPS~NSHGPFTLSGLDGPSTPGPFSPDLQKT

ll::llll

SAP-l a

complex

354

361

366

I 1111111111:11 Ill:1 381

387

IIIII

:

:

417

422

aa

:I1 III

:II

II

420

425

aa

8 1994 Current Ooinion in Genelics and Develoomenl

Fig. 1. Organization of the Ets domain proteins Elk-l and SAP-l a. (a) The polypeptide sequence is shown as a line, with the three regions of greater than 70 % conservation between Elk-l and SAP-l a shown as shaded boxes. Box A is the Ets domain, Box B is the SRF domain, and Box C contains (S/VP motifs. Recent cDNA cloning studies show that SAP-l a is 431 amino acids in length, rather than 453 as originally deduced (MA Price, R Treisman, unpublished data). (b) Conserved carboxy-terminal sequences of Elk-l and SAP-la. The carboxy-terminal regions of Elk-l and SAP-l a are shown, with vertical lines indicating sequence identities and colons indicating conservative changes. Conserved fS/T)P motifs are in bold, and Box C is indicated as boxed seq&nce.

by Ets domain truncations, such as that found in AElk1 191;AElk- can activate transcription in cotransfection studies in vivo llY.1. Three observations suggest that the only contacts between Ets proteins and SRF that are essential for ternary complex formation are made by the B box. First, ternary complex formation is largely unaffected by the nature or length of the sequences between the Ets domain and the B box [5,91. Second, binding studies with both natural SRE or synthetic DNA show that the sequence specificity of ternary complex formation is flexible, and can occur when the SRF and Ets sites are separated over a helical turn 117”l. Third, an Elk-l derivative, in which the Ets domain is replaced by the DNA-binding domain of the bacterial repressor LexA, efficiently forms SRF-dependent ternary complexes at a LexA half operator located next to an SRF-binding site 114**1. It is noteworthy that a sequence distantly related to the B box is the only region conserved among MATal proteins from different yeasts. As these proteins form ternary complexes with an SRF-related protein, MCMl 1201,this sequence may mediate MATal-MCMI interactions 121’1. Although SRF facilitates TCF binding to the c-fos SRE, SRF binding is not substantially stabilized in the ternary complex 191.Indeed, several SREs have no apparent Ets motif in their vicinity, and it remains unclear whether ternary complexes form at these SREs in vivo. It remains possible, however, that at low affinity SRF sites, SRF binds only as part of a ternary complex with TCF; this may be the case in the ntrri’7promoter 122.1.

Growth ternary

factors induce phosphorylation complex factors

of

Growth factor stimulation causes reversible phosphorylation, both of cellular TCF activities, and of recombinant Elk-l produced in transfected cells [12**,23,24”1. Moreover, intracellular activation of the MAP kinase pathway by phorbol ester treatment, or expression of activated forms of Src, Ras, Mos, and Raf, also results in phosphorylation of the Elk-l and SAP-1 carboxyl termini ([12”-14**,25”1; MA Price, R Treisman, unpublished data; M Kortenjann, H Gille, P Shaw, personal communication). The c-jun amino-terminal activation domain is also phosphorylated at (S/TX’ motifs in response to phorbol esters, UV light, and activated ~6s and SK alleles (reviewed in 1261). Nevertheless, this pathway probably differs from that leading to Elk-l, because the relative susceptibility of the two targets to these stimuli differs (127’1; J Wynne, R Treisman, unpublished data), and the Jun amino-terminal kinase is distinct from MAP kinase 128’1. Phosphopeptide fingerprints of Elk-l derivatives from cells in which the MAR kinase pathway has been activated by growth factor stimulation or oncogene expression are substantially identical to those generated from Elk-l derivatives labelled in vitro using partially purified MAP kinase; mutational analysis indicates that phosphorylation occurs at multiple (S/T)P motifs in the Elk-l carboxy-terminal region (112**-14”l; R Marais, R Treisman, unpublished data; H Gille, M Cobb, 1’ Shaw, personal communication). Sequence analysis has provided direct evidence for regulated phosphorylation

97

98

Oncogenes

and cell proliferation

of Elk-l (S/T)P motifs at serines 383, 389, and 422, and biochemical analyses indicate that modification of the other (S/T)P motifs also occurs 112**-14.01. Studies with different mutants suggest that although cooperative, each phosphorylation can occur independently of the others (112”l; H Gille, M Cobb, P Shaw, personal communication). The use of in-gel renatured kinase assays has shown that the growth factor activated Elk-l carboxy-terminal kinase has an Mr of 42 kDa 112**1, and biochemical fractionation studies indicate that Swiss 3T3 cell Elk1 kinase activity cofractionates with myelin basic protein kinase activity (H Gille, M Cobb, P Shaw, personal communication; also see 124”l). In the case of Elk-l, a simple model would be that all carboxy-terminal phosphorylations are carried out by p42/p44 MAP kinases; however, of the (S/T)P motifs in Elk-l, only those at positions 324 and 389 (SAP-l residue 387) conform to the PX(S/T)P consensus derived for p42/p44 MAP kinase 129,301 (although note that in SAP-la the two carboxy-terminal (S/T)P motifs also fit this consensus, see Fig. lb). It therefore remains possible that other kinases are also involved. Indeed, in v-raftransformed murine macrophages, it appears that MAP kinase dependent and independent pathways target Elk-l and SAP-l related TCFs, respectively (RA Hipskind, A Nordheim, M Baccarini, personal communication). Perhaps the conservation of the C box sequence reflects its modification by the same kinase(s) in all TCF family members, while the non-conserved sequences outside it are subject to differential regulation in the different proteins (see below). It is likely that dephosphorylation of ternary complex factors involves phosphatase 2A; this enzyme will dephosphorylate the proteins in vitro, and okadaic acid treatment prolongs the persistence of the phosphorylated forms of TCF in vivo 124”l. Elk-l also exhibits a basal level of phosphorylation in vivo, whether produced in logarithmically growing HeLa cells, serum-deprived NIH3T3 cells, or baculovirus-infected insect cells 112”,13”,24’*1. These phosphorylations are not growth factor regulated, and occur between the carboxyl terminus of the Ets domain and residue 307 112”,13”1. A recent in vitro study has reported that Elk-l promotes the autophosphorylation of p44mpk 1311;however, as yet there is no direct evidence for the involvement of Elk-l in such activation

in vim Phosphorylation regulates transcriptional activation by the ternary complex The carboxy-terminal region of Elk-l acts as a growth factor regulated transcriptional activation domain, whether brought to DNA by fusion with heterologous DNA-binding domains from Gal4 or LexA (112**,14**1; M Kortenjann, H Gille, 1’ Shaw, personal communication), as part of the ternary complex with SRF 113”1, or by autonomous binding of intact Elk-l (114**,181; J Wynne, R Treisman, unpublished data). In the ternary complex, SRF sequences carboxy terminal to amino acid 390 also contribute to the efficiency of transcrip-

tional activation (113”l; see also 132.1). In QT6 cells, by contrast, both SAP-l and Elk-l can constitutively activate transcription independently of the carboxy-terminal region 119.*1. Mutational analysis indicates that basal and regulated transcriptional activation requires multiple SP and TP motifs throughout the Elk-l carboxy-terminal region, including sequences outside the C box. In Elk-l, residues 383 and, to a lesser extent, 389 (SAP-l residues 381 and 387) are critical for activation; mutations at other positions have severe effects when combined, but only small effects individually, while mutations at the conserved core hydrophobic residues within the Elk-l C box also reduce regulated transcription, but not phosphorylation (112**,14**]; MA Price, R Treisman, unpublished data; M Kortenjann, H Gille, 1’ Shaw, personal communication). Modification of the carboxyterminal region, which is likely to change its conformation [12**1, might either increase its ability to interact with the basal machinery or allow displacement of inhibitory molecules, The critical importance of Elk-l residue 383 (SAP-la 381) is puzzling; perhaps the phosphorylated residue makes specific interactions with other molecules, while phosphorylation of other carboxy-terminal residues facilitates only local conformation changes. One consequence of the requirement for multiple phosphorylation of the carboxy-terminal region is that a relatively high basal level of phosphorylation can be tolerated without efficient activation occuring, effectively setting a threshold of kinase activity above which efficient activation can occur.

Is ternary

complex

formation

regulated?

The question of whether ternary complex formation at the c-fos SRE is regulated by growth factor stimulation is a matter of some dispute. Growth factor induced phosphorylation of TCFs has been reported either to be essential 13,231, or unnecessary 17,12**,24**1, for ternary complex formation by cell extract proteins in vitro. Similarly, phosphorylation of Elk-l by MAP kinase has been variously reported to be essential for ternary complex formation 1231, or only to reduce the mobility of the ternary complex in mobility-shift assays 112”,24”1. In the latter case, the mobility decrease occurs even with purified proteins. As phosphorylation increases, rather than decreases, the charge:mass ratio of the complex, it is likely to reflect phosphorylationinduced conformation changes. These discrepancies underline the difficulty inherent in extrapolation of the results of in vitro studies to the situation in vivo. Observations in vivo, however, suggest that even if ternary complex formation is regulated, growth factor stimulation is not an absolute prerequisite for ternary complex formation at the c-fos SRE. First, genomic footprinting experiments show proteins bound to the SRE Ets motif even before cells are stimulated by growth factors 1331.Second, fusion derivatives of SAP-l or Elk-l containing the constitutively active VPIG transcription activation domain can activate the SRE without growth factor stimulation 151.These ob-

Ternary

servations do not mean that exchange of different TCFs at the SRE is not regulated by growth factors, and an important task for the future will be the establishment of techniques for the identification of particular transcription factors bound to the SRE under particular conditions in vivo. Why are there multiple

A simple, but probably untenable, view holds that different TCFs either perform similar functions in different cell types, or are specific to particular SIXES.First, the Elk-l, SAP-1 and SAP-2 RNAs are present at similar relative levels in many different tissues (A Rogers, R Treisman, unpublished data), and although it was initially reported that Elk-l is expressed in a tissue-specific fashion, the protein is detectable in diverse cell types ([7,24*-l; R Treisman, unpublished data; RA Hipskind, A Nordheim, M Baccarini, personal communication). Second, both Elk-l and SAP-1 can access the c-fos SRE in vivo ([5]; A Rogers, R Treisman, unpublished data), and interact with diverse SREs in vitro [17”]. Two attractive possibilities are that different TCF proteins are either differentially linked to cellular signalling pathways and/or have different effects on transcription, thereby allowing the integration of different signals at the SRE by either passive or regulated exchange of factors; for example, MAP kinase dependent and independent pathways have been reported to target different TCFs (RA Hipskind, A Nordheim, M Baccarini, personal communication). Differential regulation could be brought about by differential modifications of sequences outside the C box: for example, serum-regulated activation by Elk-l requires sequences outside the C box that are not shared between SAP-1 or SAP-2 [12**]. Alternatively, C box phosphorylation might have different consequences in different TCFs, perhaps allowing activation or repression by different ternary complexes. Both SAP-1 and Elk-l, however, appear to work as regulated activators: exchange of their C boxes has no apparent effect, and both respond to

Receptor tyrosine kinases

factors Treisman

signals elicited by Ras, Raf, Mos, phorbol esters, whole serum, and a variety of polypeptide growth factors ([12”,25**1; MA Price, R Treisman, unpublished data). However, the studies on signalling pathways to TCFs are, as yet, by no means complete. Do ternary SRF?

TCFs?

complex

complex

factors

always

act with

It is possible that Elk-l and SAP-1 can activate transcription in the absence of the ternary complex with SRF, since both proteins can bind DNA autonomously; in transfection assays, both can activate transcription of reporter genes controlled by multiple high affinity autonomous DNA-binding sites ([14**,18,19”1; R Treisman, J Wynne, unpublished data). However, in NIH3T3 cells it is likely that the endogenous Elk-l level is insufficient to saturate such sites, since these reporters are only weakly serum-responsive in the absence of transfected Elk-l (R Treisman, J Wynne, unpublished data). As more immediateearly promoter sequences become available, it will be interesting to see whether any contain tandemly repeated Elk-l or SAP-1 autonomous binding sites, since these might be particularly active in cell types where the proteins are expressed at high levels. What is the role of the ternary transcription?

complex

in c-fos

Figure 2 presents a simple picture of the different targets for growth factor signals at the c-fos promoter, based on data derived from transfection studies. In transfection assays, mutations that block ternary complex formation block phorbol ester induction in several cell types, and stretch induction in cardiac myocytes; in several reports, however, induction by serum is unaffected (14,341; CS Hill, R Treisman, unpublished data, but see [2,3]). These mutations also partially block the transcription induced by growth factors acting via

TPA

I

PKC

I\

1 ISCF3a p91, 091 -like

MAP kinase

TCFs

Ets

SRF (CarG)

AP-1 /ATF

Fig. 2. Signalling pathways to the c-fos regulatory region. This shows the region surrounding the c-fos SRE, which is located 300 bp upstream of the transcription initiation site. Factor binding sites are shaded; sequence motifs are identified below, and cognate factors identified above the sites. The identity of the factor that binds the APl/ATF site remains unknown. Targets in the region for signals from polypeptide growth factors, protein kinase C (PKC) and whole serum are indicated.

99

100

Oncogenes and cell proliferation receptor ‘tyrosine kinases (RTKs) such as platelet-derived growth factor 1351 and colony-stimulating factor-l (C Hill, R Treisman, unpublished data). Reconstruction experiments suggest that in several of these cases, a ternary complex containing Elk-l can mediate signalling via the TCF site (C Hill, R Treisman, unpublished data). Signals Ram RTKs also activate c-fos via the sis-inducible element (SIE) B6,371located upstream of the SRE (Fig. 2; M Gyman, personal communication; CS Hill, R Treisman, unpublished data). The SIE interacts with members of the ISGF3a family [38*-41”1, and it is likely that the relative contributions of the SIE and TCF sites to activation by RTKs will vary between cell types, according to activity of the different signalling pathways. Serum stimulation can induce c-fos transcription independently of the SIE [42,431; moreover, while this induction is SREdependent, the observation that it is largely unaffected by mutations of the TCF binding site which block ternary complex formation suggests that it may include a significant TCF-independent component. If so, it will be important to identify the signalling molecules involved. Conclusions The TCF protein family constitutes a group of transcription factors whose activity is directly linked to the activity of growth factor activated signalling pathways. Future work must concentrate on attempts to integrate further our knowledge of the properties of the SRE ternary complex into studies of the intact promoters of the c-fos and other immediate-early genes. In addition, more work needs to be done to address the mechanisms by which different TCF family members interact with both upstream cellular signalling pathways and downstream events involved in transcription initiation.

Acknowledgements I thank Manuela Baccarini, Mike Gihnan, Alfred Peter Shaw for permission to quote data prior and members of my group, TII Hunt and Nit stimulating discussions and incisive comments on

Nordheim and to publication, Jones both for thii manuscript.

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GRAHAM R, GI~MAN M: Distinct Protein Targets for Signals Acting at the c-jbs Serum Rcsponsc Element. Scfence 1991, 2513189-192. DALTON S, TREISMANII: Characterization of SAP-l, a Protein Recruited by Strum Response Factor to the c-jbs Serum Rcsponsc Element. Cell 1992, 68:597-612. RAO VN, HUEBNERK, I~OBE M, AR-RUSHDIA, CROCE CM, REDDY ES: elk, Tissue-Specific e&s-Related Gcncs on Chro mosomcs X and 14 near Translocation Breakpoints. Science 1989, 244:6&70. HIPSKIND RA, mo VN. MUELLER CG, REDDY ES, NORDHUM A: E&Related Protein Elk-l is Homologous to the c-/o Rep ulatory Factor p62TCE Nature 1991, 354:531-534.

WASYLYK B, HAHN SL, GIOVANE A: The Ets Family of Transcription Factors. Errrj Bfocbem 1993, 211:7-18. Comprehensive review of the Ets protein family. JANKNECHTR, NORDHEIMA: EIk-1 Protein Domains Rcquircd 9. for Diict and SRF-Assisted DNA-Biding. Nftclefc Acfds Res 1992, 20:3317-3324. 10. RAO VN, REDDY ES: cIk-1 Domains Responsible for Autonomous DNA Binding, SRESRF Interaction and Ncgativc Regulation of DNA Binding. Oncogene 1992, 7:2335-2340. 11. JANKNECH’TR, ZINCK R, ERNSTWI-I, NORDHEIMA: Functional .. Dissection of Transcription Factor EIk-1. Oncogene 1994, 9:in press. Transient transfection experiments mapping domains of Elk-l involved in processes such as transcriptional activation and nuclear localization. 12. MARAIS R, WYNNE J, TREISMANR: The SRF Accessory Pro .. tcin Elk-1 Contains a Growth Factor-Regulated Transcrip tional Activation Domain. Cell 1993, 73:381-393. Growth factor induced and Ras-induced phosphorylation of Elk-l induces apparent change in conformation of the ternary complex. Identilication of S/T-P motifs phosphorylated In vfw and identity with those phosphorylatcd in vfrro by MAP kinase. Demonstration that multiple phosphorylations regulate transcriptional activation. HILL CS, MARAIS R, JOHN S, WYNNE J, DALTON S, TREISMAN 13. .. R: Functional Analysis of a Growth Factor-Responsive Transcription Factor Complex. Cell 1993, 73:395-406. Generation of altered specificity derivatives of SRF and Elk-l that reconstitute regulated activation by the ternary complex, and that contribute to transcriptional activation. Demonstration that only the B region mediates essential contacts with SRF In the complex. 14. JANKNECHTR, ERNST WI-I, PINGOUD V, NORDHUM A: Acti.. vation of TCF Elk-1 by MAP Kinascs. &f/30 / 1993, 12:5097-5104. Identilication of (S/T)P motifs phosphorylated In vfw and identity with those phosphorylatcd in vftro by MAP kiise. Demonstration that multiple phosphorylations regulate transcriptional activation.

reading

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1991,

8. .

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R Treisman, Transcription Laboratory, Imperial Cancer Research Fund, PO Box 123, Lincoln’s Inn Fields, London WC2A 3PX, UK.

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