Regulation of transcription factor activity during ...

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Regulation of transcription factor activity during cellular aging Keith Wheaton, Peter Atadja, and Karl Riabowol

Abstract: Several lines of evidence suggest that the limited replication potential of normal human cells is due to the presence of an intrinsic genetic programme. This "senescence programme" is believed to reduce the incidence of cancer by limiting the growth of most of the transformed cells arising in vivo, although some cells do escape senescence becoming both immortalized and transformed. Here we review the literature that describes the senescence process in terms of gene expression and the regulation of gene expression by a variety of mechanisms affecting transcription factor activity. We focus on regulation of the c-fos gene through posttranslational modification of the serum response factor (SRF) as an example of altered gene expression during cellular aging. Key words: cellular aging, transcription, Fos, SRF, phosphorylation.

Resume : Plusieurs ClCments suggkrent que le potentiel de rkplication limit6 des cellules humaines normales serait attribuable B un programme genCtique intrinskque. Ce "programme de sknescence" rkduirait l'incidence du cancer en limitant la croissance de la majorit6 des cellules transformees in vivo; cependant, quelques cellules Cvitent la ~Cnescence,sont transformCes et deviennent irnmortelles. Dans cet article, nous faisons une revue de la IittCrature se rapportant i l'expression gCnique et sa regulation par divers mecanismes modifiant I'activitC des facteurs de transcription au cours du processus de la ~Cnescence.Cette revue porte particulikrement sur la rCgulation du gkne c-fos par une modification post-traductionnelle du facteur rkactionnel skrique (SRF), un exemple d'une modification de l'expression d'un gkne au cours du vieillissement cellulaire. Mots clis : vieillissement cellulaire, transcription, Fos, facteur rCactionnel sCrique (SRF), phosphorylation. [Traduit par la ridaction]

Received May 28,1996. Revised July 23,1996. Accepted July 26, 1996. Abbreviations: SRF, serum response factor; HDF, human diploid fibroblasts; MPD, mean population doubling; kDa, kilodalton(s); CDK, cyclin-dependent lunase; PKA, CAMPdependent protein kinase; PKC, protein kinase C; CKII, casein kinase II;bHLH, basic helix-loophelix; FRA, Fos-related antigen; API, activator protein I; CRE, CAMP-response element; SIE, SIS-inducible element; SRE, serum response element; UV, ultraviolet; SOS, son of sevenless; 1,2-DAG,1,2-diacylglycerol; MAPKK, mitogen-activated protein kinase kinase; TCF, ternary complex factor; MARCKS, myristylated alanine rich C kinase substrate; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; IL2, interkleukin 2; TPA, tumor-promoting agent.

K. Wheaton, P. ~ t a d j a , 'and K. ~ i a b o w o l ?Department of Medical Biochemistry and Southern Alberta Cancer Research Centre, University of Calgary Health Sciences Centre, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada.

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Present address: Amgen Institute, 620 University Avenue, Toronto, ON M5G 2C 1, Canada. Author to whom all correspondence should be addressed. e-mail: [email protected]

.ochem. Cell Biol. 74: 523-534 (1996). Printed in Canada / Imprim6 au Canada

Introduction Replicative senescence is the culmination of a form of cellular aging that is experienced by all normal cell types capable of growth in vitro (reviewed in Goldstein 1990; Campisi 1996) and in vivo (Dimri et al. 1995). This phenomenon manifests itself as a loss of mitotic activity after cells undergo a defined number of divisions. A growing body of evidence indicates that a genetic programme contributes to, or may underlie, the transition of growth-competent cells into a state of senescence. The only way in which cells appear to be able to escape from this "senescence programme" is by the poorly understood process of immortalization. Immortalization is often linked to cellular transformation seen in many emerging cancers (DiPaolo et al. 1993) and to the activation of the enzyme telomerase, which stabilizes chromosome ends through the maintenance of telomeres (Kim et al. 1994). Human diploid fibroblasts (HDFs) are cells that help to form connective tissue in the body. HDFs also serve as a widely used model in the study of cellular senescence because they grow well in culture and have limited, but very reproducible, proliferative lifespans (Hayflick and Moorhead 1961; Hayflick 1965). As shown in


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Fig. 1. Morphology of young and old fibroblasts. Low passage (upper panel, 34 mean population doublings (MPD)) and high passage (lower panel, 82 MPD) Hs68 human diploid fibroblasts (ATCC CRL#1635) growing in complete medium were fixed and stained with 0.1 pgImL of Hoechst 33258 dye for 10 s followed by 1 pgImL of Texas Red conjugated phalloidin for 10 rnin. Conditions of cell fixation, washing, and photography were as described (Atadja et al. 19956) with the exception that double exposures were taken to simultaneously visualize the actin stress fibres stained by phalloidin and the DNA stained by Hoeschst. The bars in the lower right corners represent 50 pm.


Table 1. Transcription factor expression and activity in senescent fibroblasts. Transcription factor APl* c-fos CBPItk CREBP CTF E2F- 1 fosB HSF Id- 1 Id-2 SRF* c-fos c-jun c-myc CREBP Fra- 1 GREBP

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References

Decreased expression or activity? Atadja et al. 1994, Riabowol et al. 1992, Dimri and Campisi 1994 WI-38, IMR90, HF, A2, MRC5 & Hs68 HDFs Riabowol 1992, Phillips et al. 1992, Seshadri and Campisi 1990, WI-38 and HS68 HDFs Riabowol et al. 1992 IMR-90 fibroblasts Pang and Chen 1993 Dimri and Campisi 1994, Singh and Kanungo 1993 WI-38 & Hs68 HDFs WI-38 fibroblasts Dimri and Campisi 1994 WI-38 fibroblasts Dimri et al. 1994 WI-38 fibroblasts Lucibellow et al. 1993 Niedzwiecki et al. 1991, Heydari et al. 1993, Choi et al. 1990, Liu Animal model, IMR-90 et al. 1991 TIG-3 fibroblasts Hara et al. 1994 TIG-3 fibroblasts Hara et al. 1994 Atadja et al. 1994 Hs68, WI-38, HF & A2 HDFs No change in expression or activity Lucibello et al. 1993 Phillips et al. 1992, Lucibello et al. 1993 Lucibello et al. 1993 Atadja et al. 1994 Lucibello et al. 1993 Dimri and Campisi 1994 Dimri and Campisi 1994 Dimri and Campisi 1994 Atadja et al. 1994, Dimri and Campisi 1994

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WI-38 fibroblasts WI-38 fibroblasts WI-38 fibroblasts Animal tissue WI-38 fibroblasts WI-38 fibroblasts WI-38 fibroblasts WI-38 fibroblasts WI-38 & Hs68 HDFs

myf-5 myogenin myoD OctBP ~ 5 3 pRb* SSTBP

Increased expression or activity Animal tissue Musaro et al. 1995 Animal tissue Musaro et al. 1995 Animal tissue Musaro et al. 1995 WI-38 fibroblasts Dimri and Campisi 1994 Atadja et al. 1995a Hs68, WI-38, HF & A2 HDFs Stein et al. 1990, Futreal and Barrett 1991, Riabowol 1992 IMR90, Hs68 HDFs Hs74 fibroblasts Hubbard et al. 1995

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*Factors showing differential phosphorylation. ?Change is in activity and (or) expression in old cells compared with young cells.

Fig. 1, HDFs increase in size as they age in culture and show additional phenotypic changes including increased size of actin stress fibres and an increased proportion of cells containing irregular and multiple nuclei. Although HDFs in culture are separated from their normal cellular environment in vivo, several observations support the use of HDFs grown in vitro as a valid model of biological aging: (i) fibroblasts isolated from young individuals are reproducibly able to undergo more mean population doublings (MPDs) than cells from old donors of the same species (Martin

et. al. 1970); (ii) fibroblasts from longer-lived species undergo more population doublings than short-lived species, indicating a relationship between the maximum lifespan of a species and the proliferative capacity of their fibroblasts grown in culture (Goldstein 1974; Rohme 1981); (iii) fibroblasts from individuals with premature aging syndromes such as progeria and Werner's syndrome undergo fewer population doublings in culture than normal cells (Brown 1990); and (iv) human fibroblasts also appear to normally become senescent in vivo (Dimri et al. 1995). These and other recent observations

Biochern. Cell Biol. Vol. 74. 1996 regarding biochemical differences and changes in gene expression that accompany HDF senescence suggest that the HDF model of cellular aging in many ways reflects the normal biological processes that occur during in vivo aging.


Gene expression in aging human diploid fibroblasts Significant changes in the expression of many genes including those encoding transcription factors, cell cycle regulatory proteins, protooncogenes, tumour suppressors, structural proteins, and metabolic enzymes have been reported in aging cells (Rittling et al. 1986; Wong and Riabowol 1996; reviewed in Meyyappan et al. 1996).However, it is not known whether the altered expression of specific genes can play a causal role in the aging process or to what extent these changes contribute to the senescent phenotype. Because individual transcription factors are capable of modifying the expression of entire subsets of genes, focusing on the molecular mechanisms by which gene expression is coordinately altered should help to identify signal transduction pathways essential to the understanding of senescence. Many of the transcription factors that have been examined in senescent HDFs are listed in Table 1.

Tumour suppressor activity increases during senescence The products of two tumour suppressor genes, p53 and Rb, have been investigated by several groups in aging fibroblasts. The p53 gene encodes a nuclear phosphoprotein that is commonly mutated in a large number of cancer types (reviewed in Levine 1993) and that acts as a sequence-specific transcription factor (Kern et al. 1991).p53 negatively regulates growth by promoting the transcription of growth inhibitory genes such as the 21kDa inhibitor (p21) of cyclin-dependentkinases (CDK; Farmer et al. 1992).Intracellular levels of p53 protein are elevated after radiation-induced DNA damage, which arrests cell growth (Kastan et al. 1991; Lu and Lane 1993), and this p53 increase has been proposed to constitute part of a cell cycle checkpoint in whichp53-inducedp21 expression inhibits CDK activity and proliferating cell nuclear antigen. These changes subsequently block progression through the cell cycle until DNA repair can be completed (el-Deiry et al. 1993). Although p53 levels increase in response to DNA damage, examination of p53 mRNA and protein levels in young and old HDFs has shown that little change occurs with age (Rittling et al. 1986; Afshari et al. 1993;Atadja et al. 1 9 9 5 ~ )However, . both p53 DNA binding and transcriptional activity were found to increase with in vitro age, suggesting that an expression-independent mechanism of p53 activation occurs during cellular senescence (Atadja et al. 199.5~).Although no convincing evidence for activation of p53 binding by phosphorylationwas seen in senescent cells, recent studies have demonstrated that phosphorylation of certain amino acid residues can enhance the specific DNA binding activity of p53. For example, immunopurified human p53 that was phosphorylated in vitro by S and G2/M CDKs bound several promoters more avidly than unphosphorylated p53, including the promoter found upstream of the gene encoding the CDK inhibitor p21 (Wang and Prives 1995). Although the 105-kDa Rb protein does not appear to be a transcription factor per se, it has the ability to modulate transcription factor activity depending on its phosphorylation

state. The Rb protein is differentially phosphorylated during the cell cycle, being underphosphorylated in Go and G1 and becoming progressively more phosphorylated through late G1, S, G,, and M (reviewed by Levine 1993). The underphosphorylated form of Rb is believed to represent the active form of the protein that acts to specifically bind the E2F transcription factor, blocking its ability to transcriptionally activate positive regulators of cell cycle progression. In several independent studies, senescent and young HDFs were found to have comparable levels of Rb protein; however, senescent cells appear unable to inactivate Rb by phosphorylation in response to growth factors (Stein et al. 1990; Futreal and Barrett 1991; Riabowol 1992). This may be due to decreased levels of cyclin-CDK complexes capable of phosphorylating Rb (Stein et al. 1991; Dulic et al. 1993); however, levels of the D-type class of cyclins, which have been reported to selectively phosphorylate Rb, increase several-fold during cellular aging (Dulic et al. 1993; Atadja et al. 1995b), implying a different mechanism. A likely basis for the inability of senescent cells to phosphorylate Rb may be the dramatic increase seen in the expression of several CDK inhibitors, including p21 (Noda et al. 1994; Atadja et al. 1995b), p16, and p27 (Wong and Riabowol 1996), with increasing in vitro age. Regardless of the mechanism responsible, the constitutive activation of the potent growth inhibitors p53 and Rb likely contributes to the inability of senescent cells to respond fully to mitogens.

Modification of transcription factor activity A number of mechanisms have the potential to regulate transcription factor activity. Of these, the activation of protein kinase cascades leading to pivotal phosphorylation events has been studied closely in many cell systems, including aging HDFs (reviewed by Karin 1994). Four major means by which phosphorylation can affect transcription factor activity include (i) translocation across the nuclear membrane (Brown et al. 1995), (ii) altering DNA binding (Atadja et al. 1994, Takenaka et al. 1995), (iii) direct modulation of transactivation domain activity (Bannister et al. 1994), (iv) and multimerization (Brown et al. 1995). In some cases, expression of a particular gene can be regulated by combinations of these mechanisms converging on a single transcription factor (reviewed in Boulikas 1995). Such posttranslational modifications can have profound effects on protein conformation or on protein-protein interactions, leading to altered activity.

Nuclear translocation This regulation mechanism allows transcription factors that are sequestered within the cytoplasm to enter the nucleus in response to intracellular and extracellular signals. Typically these signals result in the activation of kinases and phosphatases leading to transcription factor phosphorylation or dephosphorylation. One example of this mechanism is found in the Rel-related family of transcription factors, many of which are phosphorylated by CAMP-dependentprotein kinase (PICA) or protein kinase C (PKC), which results in their exclusion from the nucleus (reviewed in Gilmore 1991). However, other transcription factors such as NF-AT, NFIL-6, and ISGF3 are induced to translocate across the nuclear membrane by phosphorylation (reviewed in Hunter and Karin 1992). As an additional level of regulation, protein kinases themselves can

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be translocated across the nuclear membrane. For example, PKA (Nigg et al. 1985) and MAPERK (Chen et al. 1992) protein kinases are regulated by kinase cascades that are initiated from the plasma membrane. Upon activation, PKA and MAPK translocate and are subsequently able to phosphorylate a variety of substrates, including the transcription factors CREB and Elkl, which stimulate the expression of many genes including c-fos (Riabowol et al. 1988b; Ginty et al. 1994).


DNA binding Binding of sequence-specific transcription factors to DNA can be affected either positively or negatively by phosphorylation. Generally, phosphorylation of amino acid residues just adjacent to or within the DNA binding site of the factor decreases DNA binding activity. For example, phosphorylation of residues near the DNA binding domains of c-Jun (Boyle et al. 1991) and c-Myb (Luscher et al. 1990) inhibits binding. Both of these factors have casein kinase I1 (CKII) sites that are phosphorylated by this kinase in vitro. In the cell, CKII is constitutively active, insuring that all of the transcription factors that it targets are repressed; however, activity of these factors can be restored by dephosphorylation (Luscher et al. 1990; Boyle et al. 1991). Several mechanistic possibilities exist to explain why phosphorylation inhibits binding. Phosphorylation of a transcription factor could inhibit DNA binding by inducing a conformational change that alters its affinity for a specific DNA sequence or by specifically targeting areas or residues critical in protein - DNA phosphate backbone interactions. In the latter case, resulting electrostatic repulsion would inhibit DNA binding activity. One example of this mechanism is the phosphorylation of myogenin by PKC at several sites in the binding domain, including one on a residue that is conserved in all basic helix-loophelix (bHLH) proteins (Li et al. 1992). Because this residue is conserved, it implies that there may be a common mechanism for the inactivation of many bHLH transcription factors. Stimulation of transcription factor binding activity is a less common consequence of phosphorylation than inactivation, but two factors that have been reported to bind to DNA more avidly following phosphorylation target the SRE of the c-fos gene. In the case of one of these factors (Elkl), it is clear that phosphorylation promotes binding and transcriptional activation. However, as discussed below, the specific effects of phosphorylating serum response factor (SRF; Norman et al. 1988) are less clear.

Regulation of transcriptional activation domains Transcriptional activation domains can also be phosphorylated to modulate transcription factor activity by promoting or repressing their association with other cofactors or with the basal transcriptional machinery. In most instances, phosphorylation acts to positively regulate activity by increasing the affinity of the transactivation domain for a component in the basal transcriptional machinery. For example, the bZIP family of transcription factors are phosphorylated by various kinases and induce transcriptional activation (Gonzalez et al. 1989). Specific examples from this class include CREB and c-Jun, which are targeted by PKA (Boshart et al. 1991) and MAPK (Binetury et al. 1991), respectively. Negative regulation of activity by phosphorylation is shown by such factors as ADRl and c-Fos. Evidence suggests that ADRl is inactivated by PKA phospho-

rylation, preventing it from interacting with the transcriptional machinery while DNA binding activity is unaffected (Taylor and Young 1990). Similarly, the lower amounts of c-Fos protein expressed in senescent fibroblasts are distinct highly phosphorylated isoforms that appear unable to bind c-Jun and subsequently cannot interact with their target DNA sequence (Riabowol et al. 1992). The c-Fos protein has also been reported to be able to transrepress its own transcription, and transrepression appears to depend on the phosphorylation state within specific C-terminal regions of the protein (Ofir et al. 1990).

Transcription factor expression and activity during cellular aging By preparing nucleaf extracts from young and old fibroblasts and testing their ability to bind to consensus DNA sites in mobility shift assays, many transcription factor activities have been identified that are altered in old cells (Riabowol et al. 1992; Atadja et al. 1994; Dimri and Campisi 1994; Atadja et al. 1 9 9 5 ~ )One . gene encoding a transcription factor that is repressed dramatically in senescent cells is the protooncogene c-fos (Seshadri and Campisi 1990; Winkles et al. 1990; Irving et al. 1992; Phillips et al. 1992; Riabowol 1992; Riabowol et al. 1992; Sikora et al. 1992; Atadja et al. 1994) although the extent of repression may vary widely depending on growth conditions (Lucibello et al. 1993) and background genetic mutations (Oshima et al. 1995). The c-fos gene encodes a member of a family of transcription factors consisting of cFos, FosB, and Fos-related antigens 1 and 2 (FRA1, FRA2), which, together with the members of the c-Jun family of transcription factors (c-Jun, JunB, and JunD), comprise various dimeric complexes collectively called the activator protein 1 (AP1) transcription factor. Combinations of homodimers of Jun proteins and heterodimers consisting of one Fos family member and one Jun family member form different AP1 complexes that regulate the expression of many genes containing variants of the consensus binding site TGA(G1C)TCA (Kovary and Bravo 1 9 9 1 ~ ) .The expression of c-Fos is required for cell growth in transient assays (Holt et al. 1986; Nishikura and Murray 1987; Riabowol et al. 1988a; Kovary and Bravo 1991b; Cosenza et al. 1994), for normal mouse development (Wang et al. 1992), and for malignant progression (Saez et al. 1995) but appears dispensable for cell growth in chronic assays when knocked out by homologous recombination (Field et al. 1992; Wang et al. 1992). Continuous expression of c-jos can lead to tumour induction (Lee et al. 1988) and aberrant differentiation (Muller and Wagner 1984) but can sometimes block differentiation (Ito et al. 1989).

Regulation of c-fos during senescence The expression of c-Fos is required for the growth of HDFs in short-term assays and the c-fos gene is strongly down-regulated during aging in most normal cell strains examined. The basis of this down-regulation was therefore studied in young and old HDFs by examining the activities associated with different elements of the c-fos promoter. The c-fos promoter contains a consensus TATA box, a CAMP-response element (CRE) positioned where genes often contain a CAAT box, and two additional proximal elements that have been identified as regulators of c-fos transcription (Gilman et al. 1986; Treisman 1986). These include the SIS-inducible element (SIE) and the

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Fig. 2. Regulation of c-fos expression through phosphorylation of upstream transcription factors. Ligand binding induces dimerization and autophosphorylation of growth factor receptors leading to sequential binding of Grb2, SOS, Ras, and Rafl. Localization of Rafl to the membrane and (or) stimulation of Rafl through phosphorylation by PKC initiates a kinase cascade leading to translocation of both MAPK and p9(YSkinto the nucleus, where they phosphorylate the transcription factors Elk1 and SRF, respectively.

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serum response element (SRE), the latter being both necessary and sufficient for gene expression in response to mitogenic stimulation by serum (Treisman 1986). The relative locations of these elements within the c-fospromoter and a major signal transduction pathway that impinges on regulation of the c-fos gene through mitogens is shown in Fig. 2. The SRE mediates c-fosactivation by many stimuli, including serum, PDGF, EGF, cytokines (IL2), phorbol esters (TPA), UV light, ca2+,wounding, oxidants, and antioxidants. One of the signal transduction pathways of growth factor stimulated c-fos expression starts with dimerization of growth factor receptors after ligand binding, leading to autophosphorylation of residues within the cytoplasmic domains of the receptors (Fantl et al. 1993). This, in turn, is followed by SH2 domain containing molecules interacting with the receptor and recruiting other factors including adaptors. One such adaptor molecule is Grb2, which contains both SH2 and SH3 domains, which bind to the growth factor receptor and to the son of sevenless (SOS) protein, respectively. The SOS guanine exchange factor catalyses the transformation of inactive GDP-ras to active GTP-ras (Boguski and McCormick 1993) and activated Ras then recruits Raf 1 to the membrane, leading to its activation (Moodie and Wolfman 1994). Rafl is also activated via phosphorylation by PKC, and because PKC is dependent on, and activated by, 1,2-diacylglycerol (DAG), c-fosis induced by the DAG analog TPA. Appropriately localized and activated Rafl then initiates a kinase cascade through phosphorylation of mitogen-activated protein kinase kinase (MAPKK) that culminates in the phosphorylation of ternary complex factors (TCFs) and SRF as outlined in Fig. 2. Following the rapid stimulation of c-fos transcription by mitogens, its expression is then rapidly down regulated and this attenuation coincides with dephosphorylation of TCF. Consistent with the idea that inactivation of TCF may be responsible for the down-regulation of c-fos, addition of phosphatase inhibitors blocks both the dephosphorylation of Elk1 and the down-regulation of c-fos transcription following serum stimulation (Zinck et al. 1993, 1995). Phosphorylation of the C-terminal domain in Elkl is now well correlated with the ability of TCF to interact with SRF (Gille et al. 1992) and to allow transactivation (Gille et al. 1995) in that phosphorylation of serine 383 of Elkl is directly related to its ability to form a quaternary complex comprised of two SRF and two Elkl molecules assembled onto the SRE (Gille et al. 1996). Despite the fact that phosphorylation of TCFs such as Elk1 has been shown to promote and to be necessary for activation of the c-fosgene, the effects that the phosphorylation state of SRF has on transcription are less obvious. For example, phosphorylation of SRF by CKII has been reported to enhance DNA binding (Manak et al. 1990) or to stimulate the on-off rates of the transcription factor with its target DNA sequence in the absence of increased binding affinity (Marais et al. 1992; Janknecht et al. 1992; Rivera et al. 1993), but this phosphorylation does not appear to effect the expression of the cfos gene (Janknecht et al. 1992; Manak and Prywes 1993). In contrast, the increased phosphorylation of SRF observed to occur in vivo in senescent HDFs correlates very strongly with the inability of SRF from old cells to bind to the c-fos SRE (Atadja et al. 1994), perhaps accounting for the repression of c-fos transcription during cellular aging. Because previous

studies have reported that phosphorylation of SRF by other kinases can activate DNA binding (Rivera et al. 1993) or promote transactivation (Liu et al. 1993; Miranti et al. 1995), the inhibition of SRF by hyperphosphorylation may represent a mechanism unique to the aging of normal diploid cells. However, analysis of this effect must include data regarding the specific amino acid residues targeted and the kinase(s) potentially involved.

SRF: structure and regulation of activity As shown in Fig. 3, the SRF protein can be defined as consisting of three functionally distinct domains: the N-terminal domain, the MADS box DNA binding and dimerization domain, and the C-terminal domain. The domains outside the MADS box act primarily to modulate the binding activity and the transcriptional activation ability of the protein. As can be seen from Fig. 3, the 508 amino acid SRF protein is very rich in phosphorylatable residues consisting of 12.6% (641508) serine, 11.6% (591508) threonine, and 1.8% (91508) tyrosine, making the protein a prime candidate for regulation through phosphorylation. The N-terminal domain of human SRF plays a significant role in imparting DNA binding specificity (Sharrocks et al. 1993). Several phosphorylation sites in this domain have been reported to be necessary for maximal DNA binding activity while others appear to alter the on-ff rate of SRF with its cognate DNA sequence. Although the constitutive phosphorylation of amino terminal residues by CKII is not thought to be significant in mediating transcriptional induction in response to stimuli (Janknecht et al. 1992; Manak and Prywes 1993), growth factor regulated phosphorylation by the ribosomal S6 kinase pp90rSkon serine 103 has been reported to affect the affinity and rate with which SRF associates with its binding site (Rivera et al. 1993). The C-terminus is the transcriptional activation domain of SRF and functions both in vivo and in vitro. Maximal response to TCF-dependent and -independent pathways of cfos transcriptional induction require the function of the C-terminal domain of SRF. This region is not rich in any particular amino acid and does not contain a large negative charge and, therefore, does not belong to any standard classes of activation domains (Shore and Sharrocks 1995). This domain is thought to interact with the TFIIF portion of the transcriptional machinery (Zhu et al. 1994), and interaction with the RAP74 subunit of TFIIF has been reported to be required for transcriptional activation by SRF (Joliot et al. 1995). Phosphorylation of specific residues by the DNA-activated serinel threonine-specific protein kinase (DNA-PK) has also been noted in this region at serines 435 and 446 (Liu et al. 1993). Amino acid substitutions that disrupted phosphorylation by DNA-PK in this region reduced SRF's ability to transactivate. DNA-PK also phosphorylates other transcription factors such as c J u n and the tumour suppressor p53; however, its precise role in the regulation of SRF and these other factors is unclear. The 90 amino acid core region of SRF (comprised of amino acids 133-222 as shown in Fig. 3) functions in dimerization and in sequence specific DNA recognition. Most of this region consists of a 57 amino acid motif called the MADS box, named for four of the originally defined members (MCM-1 Agamous-Deficiens-SRF) of a group of related transcription

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Fig. 3. Primary structure of the SRF protein. Amino acid residues within the MADS box are highlighted in grey, and the potentially phosphorylatable amino acids serine, threonine, and tyrosine are indicated in red, blue, and green, respectively.


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factors that bind a CC(FL/T)~GGconsensus sequence found within several promoters including the SRE of c-fos (reviewed in Shore and Sharrocks 1995). Biochemical studies indicate that the N-terminal portion of the MADS box is required for DNA binding specificity while the carboxyl half of the MADS box is required for dimerization. Additionally, the SRF MADS domain recruits other transcription factors such as Elk1 into a multicomponent regulatory complex (de Belle et al. 1991) with significant consequences to transcriptional regulation. The crystal structure of the SRF MADS box domain (residues 132 to 223) bound to its consensus 5equence as a dimer has been resolved to a resolution of 3.2 A (1 A = 0.1 nm) for SRF (Pellegrini et al. 1995). The solved structure reveals how the multiple functions of DNA binding, dimerization, and recruitment of collaborating factors are integrated into a single protein domain. Interaction with DNA is mediated primarily by several pairs of basic residues (lysine or arginine) that straddle the phosphodiester backbone as well as by additional electrostatic and van der Waals forces. Threonine 159 and serine 162 make hydrophobic contact with the DNA binding site and threonine 160 forms a hydrogen bond with a phosphate moiety in the consensus binding site. In addition, the serine and threonine residues that contact the DNA, as shown in the crystal structure, are potential targets or are near targets of such kinases as PKA and PKC (see Fig. 3). Previous mutagenesis studies indicated that these residues are important in regulating the binding of SRF to DNA because mutation of these residues critically reduces binding activity (Sharrocks et al. 1993 ).

Changes in kinase activity during cellular senescence Consistent with the reports mentioned above, when total protein extracts from the nuclei of young and senescent HDFs were visualized by autoradiography, many differentially phosphorylated bands were identified (Atadja and Riabowol 1996). While changes in the activities of both kinases and phosphatases are likely to contribute to the altered levels of phosphorylated proteins, only changes in the activities of kinases have been reported to occur during cellular aging. For example, altered PKC activity has been observed in senescent fibroblasts where a change in serum-induced translocation of PKC from the cytosol to the cytoplasmic membrane was reported (De Tata et al. 1993). In contrast, exogenous PMA stimulated this translocation identically in young and old cells, implying that the difference in response to serum was due to changes in the production of the enzyme effector 1,2-DAG (De Tata et al. 1993). Although old fibroblasts have a higher level of 1,2DAG (Venable et al. 1994; Vannini et al. 1994), the increases resulting from serum induction were reported to be much lower in senescent cells (Chang and Haung 1994). This could contribute to the senescent phenotype in several ways. For example, decreased phosphorylation of substrates that are downstream of PKC could affect the expression of many growth-regulatory genes such c-fos (see Fig. 2) or other MAPK substrates. Additionally, altered PKC activity might be expected to affect the phosphorylation state of a major substrate called myristylated alanine rich C kinase substrate (MARCKS), a filamentous actin crosslinking protein with altered phosphorylation that could play a role-in the age-

related modification of the actin stress fibre component of the cytoskeleton, shown in Fig. 1.

Summary Replicative senescence is accompanied by a variety of cellular changes, some of which appear to contribute to the loss of growth potential seen during cellular aging (reviewed in Goldstein 1990; Campisi 1996). Of these changes, the elaboration of a "senescence programme" responsible for blocking continued growth is appealing because reduced expression of many positive growth regulators such as c-Fos, E2F, PCNA, and CDKl and increased expression of growth inhibitors such as the p16 and p21 CDK inhibitors and genes involved with differentiation such as myoD and cyclin D l have been reported by many groups (reviewed in Meyyappan et al. 1996). Because an age-related modification of transcription factor activity has the potential to impinge upon the expression of a large number of genes, the regulation of the c-fos gene was investigated and it was found that decreased expression was due to the loss of SRF binding activity (Atadja et al. 1994). Strong evidence was obtained indicating that hyperphosphorylation of SRF was responsible for the age-related decline in c-fos expression, raising the possibility that hyperphosphorylation of additional transcription factors may underlie their altered activity, as in the case of increased p53 activity in senescent human fibroblasts (Atadja et al. 1995~).

Acknowledgements This work was supported by grants to KR from the National Cancer Institute of Canada, the Medical Research Council, and the Alberta Cancer Board. KW was the recipient of Medical Biochemistry Departmental stipend support and PA is an MRC Fellow.

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