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• W2132038146 abstract "Apoptosis involves the cessation of cellular processes, the breakdown of intracellular organelles, and, finally, the nonphlogistic clearance of apoptotic cells from the body. Important for these events is a family of proteases, caspases, which are activated by a proteolytic cleavage cascade and drive apoptosis by targeting key proteins within the cell. Here, we demonstrate that serum response factor (SRF), a transcription factor essential for proliferative gene expression, is cleaved by caspases and that this cleavage occurs in proliferating murine fibroblasts and can be induced in the human B-cell line BJAB. We identify the two major sites at which SRF cleavage occurs as Asp245 and Asp254, the caspases responsible for the cleavage and generate a mutant of SRF resistant to cleavage in BJAB cells. Investigation of the physiological and functional significance of SRF cleavage reveals that it correlates with the loss of c-fos expression, whereby neither SRF cleavage fragment retains transcriptional activity. Moreover, the expression of a noncleavable SRF in BJAB cells suppresses apoptosis induced by Fas cross-linking. These results suggest that for apoptosis to proceed, the transcriptional events promoting cell survival and proliferation, in which SRF is involved, must first be inactivated. Apoptosis involves the cessation of cellular processes, the breakdown of intracellular organelles, and, finally, the nonphlogistic clearance of apoptotic cells from the body. Important for these events is a family of proteases, caspases, which are activated by a proteolytic cleavage cascade and drive apoptosis by targeting key proteins within the cell. Here, we demonstrate that serum response factor (SRF), a transcription factor essential for proliferative gene expression, is cleaved by caspases and that this cleavage occurs in proliferating murine fibroblasts and can be induced in the human B-cell line BJAB. We identify the two major sites at which SRF cleavage occurs as Asp245 and Asp254, the caspases responsible for the cleavage and generate a mutant of SRF resistant to cleavage in BJAB cells. Investigation of the physiological and functional significance of SRF cleavage reveals that it correlates with the loss of c-fos expression, whereby neither SRF cleavage fragment retains transcriptional activity. Moreover, the expression of a noncleavable SRF in BJAB cells suppresses apoptosis induced by Fas cross-linking. These results suggest that for apoptosis to proceed, the transcriptional events promoting cell survival and proliferation, in which SRF is involved, must first be inactivated. immediate early serum response element serum response factor ternary complex factor signal transducers and activators of transcription polymerase chain reaction phosphate-buffered saline polyacrylamide gel electrophoresis 3-[(3-cholamidopropyl)- dimethylammonio]-1-propanesulfonic acid 4′,6′-diamidino-2-phenylindole double mutant benzyloxycarbonyl- Val-Ala-Asp Cell growth, proliferation, and differentiation are regulated by numerous, diverse, extracellular signals that modulate gene expression. The proto-oncogene c-fos represents a classic example of an immediate early (IE)1 gene, induced in response to growth factors and other mitogenic stimuli (1Rivera V.M. Greenberg M.E. New Biol. 1990; 2: 751-758PubMed Google Scholar, 2Shaw P.E. Gille H. Herrlich P. Angel P. The FOS and JUN Families of Transcription Factors. 1st Ed. CRC Press, Inc., Boca Raton, FL1994: 71-85Google Scholar, 3Janknecht R. Immunobiology. 1995; 193: 137-142Crossref PubMed Scopus (34) Google Scholar). Contained within the promoter of c-fos and of other IE genes is the serum response element (SRE), which is essential for strict transcriptional control. Serum response factor (SRF) is a ubiquitously expressed, 67-kDa protein that binds, as a dimer, to the central element of the SRE, the CArG box (CC(A/T)6GG) (4Johansen F.E. Prywes R. Biochim. Biophys. Acta Rev. Cancer. 1995; 1242: 1-10Crossref PubMed Scopus (105) Google Scholar). SRF belongs to the MADS box family of transcription factors, named after four proteins identified with a common structural domain (MCM1, Agamous, Deficiens, SRF), all of which regulate transcription via the recruitment of auxiliary factors (5Schwarz-Sommer Z. Huijser P. Nacken W. Saedler H. Sommer H. Science. 1990; 250: 931-936Crossref PubMed Scopus (648) Google Scholar, 6Pellegrini L. Song T. Richmond T.J. Nature. 1995; 376: 490-498Crossref PubMed Scopus (297) Google Scholar). The conserved core domain of SRF is sufficient for protein dimerization, DNA binding, and recruitment of ternary complex factors (TCFs). The TCFs form a subset of the ets family of proteins and include Elk-1, Sap1, and Net/Sap2/Erp (7Wasylyk B. Hagman J. Gutierrez Hartmann A. Trends Biochem. 1998; 23: 213-216Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). In response to activation of the mitogen-activated protein kinase signaling pathway, TCFs are rapidly phosphorylated, resulting in a dramatic increase in their transactivation potential (8Kortenjann M. Shaw P.E. Crit. Rev. Oncogen. 1995; 6: 99-115PubMed Google Scholar, 9Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1160) Google Scholar, 10Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2274) Google Scholar).In addition to its role at the SRE, SRF has been shown to regulate transcription in a TCF-independent manner. The exact function of SRF in this mode of gene expression is unclear. Within SRF, several amino-terminal phosphorylation sites have been identified (11Manak J.R. deBisschop N. Kris R.M. Prywes R. Genes Dev. 1990; 4: 955-967Crossref PubMed Scopus (118) Google Scholar, 12Manak J.R. Prywes R. Mol. Cell. Biol. 1991; 11: 3652-3659Crossref PubMed Scopus (72) Google Scholar, 13Janknecht R. Hipskind R.A. Houthaeve T. Nordheim A. Stunnenberg H.G. EMBO J. 1992; 11: 1045-1054Crossref PubMed Scopus (108) Google Scholar, 14Marais R.M. Hsuan J.J. McGuigan C. Wynne J. Treisman R. EMBO J. 1992; 11: 97-105Crossref PubMed Scopus (128) Google Scholar). More specifically, it has been demonstrated that phosphorylation of SRF at Ser103 correlates with c-fos expression (15Rivera V.M. Miranti C.K. Misra R.P. Ginty D.D. Chen R.-H. Blenis J. Greenberg M.E. Mol. Cell. Biol. 1993; 13: 6260-6273Crossref PubMed Scopus (231) Google Scholar,16Miranti C.K. Ginty D.D. Huang G. Chatila T. Greenberg M.E. Mol. Cell. Biol. 1995; 15: 3672-3684Crossref PubMed Scopus (197) Google Scholar). A carboxyl-terminal transactivation domain has also been mapped and shown to interact with components of the basal transcription machinery, in particular with Rap74, the large subunit of TFIIF (17Zhu H. Joliet V. Prywes R. J. Biol. Chem. 1994; 269: 3489-3497Abstract Full Text PDF PubMed Google Scholar,18Joliet V. Demma M. Prywes R. Nature. 1995; 373: 632-635Crossref PubMed Scopus (79) Google Scholar). TCF-independent transcriptional activation by SRF is regulated by the Rho family small GTPases RhoA, Rac1, and Cdc42hs (19Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1199) Google Scholar). These small G-proteins regulate several cytoskeletal processes, and recent data have suggested that the convergence of LIM kinase and RhoA signaling, via actin treadmilling, results in the transcriptional activation of some SRF-dependent genes (20Sotiropoulos A. Gineitis D. Copeland J. Treisman R. Cell. 1999; 98: 159-169Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar). The mechanism by which this occurs has not yet been characterized in detail, and RhoA appears to function independently of Rac1 and Cdc42hs, implying that additional signaling pathways may be involved. The CArG box is frequently found in the promoters of muscle-specific genes. In this context, it has been demonstrated that SRF is essential for myogenic differentiation. Inhibition of SRF by microinjection of anti-SRF antibodies or the expression of antisense SRF RNA repressed muscle marker gene expression and blocked the differentiation of myoblasts to myotubes (21Soulez M. Rouviere C.G. Chafey P. Hentzen D. Vandromme M. Lautredou N. Lamb N. Kahn A. Tuil D. Mol. Cell. Biol. 1996; 16: 6065-6074Crossref PubMed Scopus (82) Google Scholar, 22Gauthier-Rouviere C. Vandromme M. Tuil D. Lautredou N. Morris M. Soulez M. Kahn A. Fernandez A. Lamb N. Mol. Biol. Cell. 1996; 7: 719-729Crossref PubMed Scopus (73) Google Scholar). Moreover, homozygous SRF−/− mouse embryos fail to develop mesoderm (23Arsenien S. Weinhold B. Oelgeschläger M. Rüther U. Nordheim A. EMBO J. 1998; 17: 6289-6299Crossref PubMed Scopus (305) Google Scholar).Apoptosis, or programmed cell death, is the process by which surplus or potentially harmful cells are removed from an organism in a nonphlogistic manner. Loss of the tight control governing this process can result in inflammation, cancer, stroke, and many neurodegenerative disorders. Caspases, or cysteine-dependent aspartate-directed proteases, form an integral part of the apoptosis machinery (24Earnshaw W.C. Martins L.C. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2428) Google Scholar). In response to some extracellular stimuli, such as Fas ligand, TNFα, or genotoxic agents, caspases are activated in a hierarchical cleavage cascade. Once activated, they target and inactivate numerous intracellular substrates, including structural components of the cytoskeleton, DNA repair enzymes, protein kinases, and transcription factors as stricken cells are disassembled and removed by phagocytosis (25Savill J. Nature. 1998; 392: 442-443Crossref PubMed Scopus (207) Google Scholar).It is also known that caspases have additional roles beyond apoptosis, in line with their initial identification as relatives of the interleukin-1β-converting enzyme (26Enari M. Hug H. Nagata S. Nature. 1995; 375: 78-81Crossref PubMed Scopus (797) Google Scholar, 27Los M. Van de Craen M. Penning L.C. Schenk H. Westendorp M. Baeuerle P. Dröge W. Krammer P.H. Fiers W. Schulze-Osthoff K. Nature. 1995; 375: 81-83Crossref PubMed Scopus (645) Google Scholar). For example, cleavage of GATA-1, mediated by caspases, has recently been demonstrated to block erythroid differentiation (28De Maria R. Zeuner A. Eramo A. Domenichelli C. Bonci D. Grignani F. Srinivasula S.M. Alnemri E.S. Testa U. Peschle C. Nature. 1999; 401: 489-493Crossref PubMed Scopus (341) Google Scholar). In addition, caspases have been shown to cleave the transcription factors NF-κB, resulting in the down-regulation of NF-κB-dependent transcription, and STAT1, suggesting a role for caspases in mediating transcriptional responses (29Ravi R. Bedi A. Fuchs E.J. Bedi A. Cancer Res. 1998; 58: 882-886PubMed Google Scholar, 30van Antwerp D.J. Martin S.J. Verma I.M. Green D.R. Trends Cell Biol. 1998; 8: 107-111Abstract Full Text PDF PubMed Scopus (334) Google Scholar, 31King P. Goodbourn S. J. Biol. Chem. 1998; 273: 8699-8704Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar).Here we show that SRF also serves as a target for caspases in several cell types. Two adjacent cleavage sites recognized by caspases 3 and 7 allow SRF to be cleaved into two major fragments. Expression of the corresponding fragments reveals that they are unable to activate SRE-dependent transcription. Moreover, expression of a noncleavable SRF mutant suppresses apoptosis. Our results suggest that SRF is a focal point for cross-talk between signals promoting cell survival and proliferation and those promoting cell death.RESULTSThe experiments described here follow on from our initial observation of SRF fragments in NIH3T3 cells transiently transfected with an expression vector for the protein. Two polyclonal antibodies, one raised against the amino-terminal portion of SRF (α1122) and the other against the carboxyl terminus (α1795), each recognized protein fragments of 30–35 kDa, approximately half the size of full-length SRF (Fig. 1, a and b,lane 1). To gain a first indication of the nature of these fragments, we compared their size and immunoreactivity with truncated forms of SRF expressed from suitably engineered vectors. Thus, the SRF fragment recognized by α1122 was larger than Ncore (lane 5) but almost identical in size to Ncore245 (lane 4), suggesting that it corresponds to SRF amino acids 1–245. Likewise, the SRF fragment recognized by α1795 was somewhat smaller than coreC (Fig. 1,lane 3) but very similar in size to SRF-C (amino acids 251–508). These observations suggest that in NIH3T3 cells SRF is cleaved in half under certain conditions and that the fragments are stable enough to accumulate in the cells.Variations in the level of SRF fragments indicated that SRF cleavage might be a regulated event, influenced perhaps by the growth state of the cells. We also noticed a cluster of six aspartate residues in the middle of SRF. In this context, caspases, a group of aspartate-directed proteases activated in cells undergoing apoptosis, are known to target numerous key proteins as the process unfolds. To assess a potential role for caspases in the cleavage of SRF, transfected NIH3T3 cells were treated with staurosporine for various times in the presence or absence of ZVAD, to drive cells into apoptosis and inhibit caspase activity, respectively. As seen in Fig. 1, c and d, staurosporine caused an increase in the yield of SRF fragments after 6 and 12 h (compare lanes 2 and 4with lane 1), which was strongly counteracted by ZVAD treatment (lanes 3 and 5), implicating caspases in this phenomenon. In this gel, it is also apparent that each antibody detects two SRF fragments, indicating that SRF is cleaved at two or more sites by caspases active in NIH3T3 cells.In parallel analyses, NIH3T3 cells were found to enter apoptosis with delayed and heterogeneous kinetics in response to staurosporine (result not shown). However, a more efficient means of inducing apoptosis by ligation of Fas proved to be impossible, since flow cytometric analysis revealed low surface expression of the receptor on NIH3T3 cells and a corresponding failure of the antibody (Jo-2) to elicit apoptosis (not shown). For this reason, we chose to analyze caspase cleavage of SRF further in a more appropriate cell system.The human mature B cell line BJAB expresses high levels of Fas on the cell surface and can be induced to undergo apoptosis upon receptor cross-linking by the agonistic monoclonal antibody CH11 (41Nagata S. Golstein P. Science. 1995; 267: 1449-1456Crossref PubMed Scopus (3965) Google Scholar). Thus, 4 h after treatment with CH11, >50% of BJAB cells have entered apoptosis, as assayed by DAPI staining and cell counting (see Fig.2 a), and by 10 h this number has risen to >90%. To determine if this process induces SRF cleavage, lysates were prepared from BJAB cells either untreated or incubated for various times with CH11. Cleavage of endogenous SRF was assayed by immunoblotting. Four hours after CH11 cross-linking, SRF fragments of approximately 30 kDa are detected and persist for the next 6 h (Fig. 2 b, lanes 4–6,arrowheads). The appearance of these bands is blocked by ZVAD (lane 7), indicating that they are caspase cleavage products. However, very little decrease in the amount of full-length SRF is apparent. The prominent 38-kDa species seen in untreated cells are unrelated to SRF, since they are not detected by antibodies specific to the carboxyl terminus of SRF (not shown), nor are they detected in a DNA-binding assay (see below).Figure 2Induction of SRF cleavage by Fas ligation on B cells. a, human BJAB cells, grown under normal conditions, were treated with the agonistic anti-Fas antibody CH11 alone or in the presence of ZVAD. Cells were harvested at the times indicated and analyzed for apoptosis by DAPI staining. Data show results from two experiments with duplicate points. b, as in a except that cells were lysed and analyzed for cleavage of endogenous SRF by immunoblotting (IB) with an amino-terminal SRF antibody (α1122). c, extracts prepared from BJAB cells, treated with CH11 for the times indicated, were incubated with a radiolabeled DNA fragment corresponding to the c-fos SRE alone (lanes 2-9) or in the presence of antibodies specific for the amino-terminal (lanes 10–12) or carboxyl-terminal (lanes 13–15) domain of SRF. The extract from untreated cells was also incubated with the corresponding preimmune sera (lanes 16 and17). Complexes were resolved by electrophoresis on a native 5% polyacrylamide gel. The complex labeled with an arrowcorresponds to the SRF homodimer; the arrowheads indicate complexes containing SRF cleavage fragments. The identity of the complex indicated with an asterisk is unclear, but it is not recognized by any of the anti-SRF antibodies. d, a BJAB cell clone overexpressing His-tagged SRF (lanes 3 and4) and a control clone (lanes 1 and2) were untreated (−) or incubated with CH11 for 10 h (+). Cells were lysed and analyzed by SDS-PAGE and immunoblotting with an amino-terminal SRF antibody (α1122). Full-length SRF-Chis and its cleavage fragments are indicated with thin andthick arrows, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The integrity of endogenous SRF in CH11-treated BJAB cells was also investigated by electrophoretic mobility shift assay with a radiolabeled DNA probe corresponding to the human c-fos SRE (Fig. 2 c). The intensity of the SRF dimer complex is reduced by 4 h and continues to decrease up to 10 h (arrow). Moreover, two faster migrating bands appear at 2 h and decay over the same period (lower arrowheads). All three of these complexes are supershifted by α1122 (lanes 10–12), which recognizes the amino terminus of SRF, while α1795 supershifts only the upper two complexes (lanes 13–15). The simplest interpretation of these observations is that the lower complex corresponds to a homodimer of amino-terminal SRF fragments, the DNA-binding domain (amino acids 133–222) residing in the amino-terminal half of SRF, while the intermediate complex is a heterodimer in which just one SRF molecule is truncated.The apparent persistence of full-length SRF in BJAB cells seen in Fig.2 b is inconsistent with the loss of SRF-SRE complexes (Fig.2 c). To resolve this inconsistency, we generated a stable cell line that overexpresses a His-tagged version of SRF. When these cells were treated with CH11 and analyzed for SRF cleavage (Fig.2 d), we observed almost complete cleavage of SRF-Chis after 10 h (lane 4), in line with the loss of DNA binding. We also observed a nonspecific band migrating slightly faster than SRF-Chis in all lanes, which we believe co-migrates with endogenous SRF and masks its disappearance in Fig. 2 b. From these observations, we infer that SRF is completely processed in apoptotic BJAB cells.SRF cleavage was also observed in vitro when35S-labeled proteins were incubated in apoptotic BJAB cell lysates. As shown in Fig. 3 b, incubation of SRF with lysates from CH11-treated cells (lane 3), but not from untreated cells (lane 2), led to the appearance of four SRF fragments of around 35 kDa, corresponding in size to those detected in NIH3T3 cells.Figure 3SRF cleavage by recombinant caspases. a, sequence of SRF amino acids 222–271 with aspartate residues highlighted with asterisks. SRF mutants used in this study are also indicated. Below is shown a diagrammatic representation of SRF cleavage and the resultant pattern of fragments resolved by SDS-PAGE. b, recombinant SRF (lanes 1–3) or the mutants EATA (lanes 4 and5), SASA (lanes 6 and 7), and the double mutant DM (lanes 8 and9), expressed and labeled with [35S]methionine by cell-free translation, were incubated with buffer B, extracts of untreated BJAB cells (−), or cells treated for 10 h with CH11 (+). Subsequently, reactions were resolved on a 12.5% SDS-polyacrylamide gel. c, recombinant SRF, expressed and labeled with [35S]methionine by cell-free translation, was incubated alone (lane 1) or with recombinant caspase 1 (lane 2), caspase 3 (lane 3), caspase 4 (lane 4), caspase 6 (lane 5), caspase 7 (lane 6), caspase 8 (lane 7), caspase 9 (lane 8), or caspase 10 (lane 9). Subsequently, reactions were resolved on a 12.5% SDS-polyacrylamide gel. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The data shown in Fig. 1 indicate that SRF cleavage occurs in the middle of the molecule, carboxyl-terminal to the DNA binding domain, referred to as coreSRF (42Schröter H. Mueller C.G.F. Meese K. Nordheim A. EMBO J. 1990; 9: 1123-1130Crossref PubMed Scopus (91) Google Scholar). The amino acid sequence of this region of SRF (amino acids 222–271) is depicted in Fig.3 a. Within this 10% of SRF, there are six aspartate residues, of which several could constitute sites of caspase cleavage. Within the remainder of SRF, there are only six additional aspartates. Mutations were therefore introduced into SRF to remove two possible caspase consensus sites at aspartate residues 245 and 254. Both individual mutants (EATA245 and SASA254) and the double mutant (DM) were then tested for cleavage by caspases present in CH11-treated BJAB cell lysates. Each single site mutant caused the loss of two SRF fragments (Fig. 3 b, compare lanes 7and 5 with lane 3), while the double mutant abolished SRF cleavage in this assay. A similar set of results was obtained when lysates from etoposide-treated Jurkat cells were used (not shown), whereby cleavage was inhibited by the inclusion of ZVAD in the assay. Moreover, single alanine substitutions of Asp245and Asp254 had exactly the same effect as the EATA and SASA mutations on SRF cleavage in vitro (result not shown). Taken together, these results demonstrate that SRF is cleaved at aspartates 245 and 254 by caspases induced in apoptotic BJAB cells and active to varying degrees in proliferating NIH3T3 cells. Furthermore, we interpret the results to indicate that SRF can be cleaved at either but not both sites.To establish which caspase or caspases are responsible for SRF cleavage, radiolabeled SRF was incubated with each of eight active recombinant caspases and then analyzed by SDS-PAGE. This assay reveals that SRF is cleaved most effectively by caspases 3 and 7 but also, to a lesser extent, by caspases 6, 8, and 9 to yield similar fragments, suggesting that all five proteases recognize the same sites (Fig.3 c). In contrast, caspases 1 and 4 cleave SRF weakly to generate a distinct pattern of SRF fragments, while recombinant caspase 10 does not cleave SRF at all.SRF cleavage by caspases was analyzed further by comparing the susceptibility of SRF mutants to a subset of recombinant caspases (Fig.4). Caspase 1 cleaved each of the three mutants similarly to wtSRF, indicating that it does not recognize the sites at Asp245 and Asp254 but does recognize other sites in the protein. Cleavage of SRF by caspases 3 and 7 is influenced by both mutations. The mutations at Asp245 (EATA) result in the loss of two caspase 3 cleavage fragments and appear to enhance the yield of the others. The same mutant markedly reduces SRF cleavage by caspase 7. In contrast, mutations at Asp254 (SASA) lead to the reciprocal pattern of fragments obtained with caspase 3 but have little effect on SRF cleavage by caspase 7. The double mutant is resistant to cleavagein vitro by caspase 7 and is cleaved only weakly by caspase 3. Moreover, the fragment sizes suggest that caspase 3 may cleave DM-SRF at a site that is not recognized in wtSRF. In summary, caspase 7 cleaves at Asp245, while caspase 3 cleaves predominantly at Asp254 but also, suboptimally, at Asp245 and an additional site (possibly Asp261).Figure 4Identification of caspase cleavage sites. a, recombinant SRF, expressed and labeled with [35S]methionine by cell-free translation, was incubated alone (lane 1) or with caspase 1 (lane 2), caspase 3 (lane 3), caspase 7 (lane 4), or caspase 9 (lane 5), after which the reactions were resolved on a 12.5% SDS-polyacrylamide gel. b, as in a except with the mutant EATA-SRF. c, as in a except with the mutant SASA-SRF. d, as in a except with the mutant DM-SRF.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We surmised that the cleavage of SRF by caspases was likely to alter the expression of IE genes such as c-fos. Accordingly, we examined the inducible level of Fos protein in BJAB cells at increasing times after CH11 treatment. Cells were pretreated with CH11 for various lengths of time and then with phorbol ester to induce c-fosexpression, which was assayed at the protein level by immunoblotting. Phorbol ester alone induced robust Fos expression (Fig.5 a, lane 2), but prior treatment with CH11 for 1 h or longer reduced and finally abolished inducible Fos expression (lanes 3–7). Thus, Fas cross-linking rapidly leads to the inhibition of Fos protein expression. To ascertain if the loss of Fos protein could be due to proteolysis, radiolabeled Fos protein was incubated in apoptotic BJAB cell lysates. However, no degradation of Fos was observed under conditions in which SRF cleavage was apparent (result not shown). Therefore, the absence of Fos in CH11-treated cells is probably due to loss of expression. To assess the extent to which this effect was due to transcriptional inhibition, the effect of CH11 on the expression of a c-fos reporter was measured. As shown in Fig. 5 b, CH11 inhibited c-fos reporter expression by 60%, and this inhibition could be blocked by ZVAD. In contrast, a c-fos reporter from which the SRE had been deleted (F10H) was refractory to CH11 treatment. It should be noted that expression of the control gene against which transfection efficiency was normalized was also insensitive to CH11 treatment. These results suggest that IE promoters containing SREs are down-regulated early in apoptosis, while other promoters remain unaffected.Figure 5Inhibition of c-fosexpression in apoptotic cells. a, human BJAB cells, cultured for 24 h in low serum (lane 1), were treated directly with phorbol ester (TPA) (lane 2) or after pretreatment with CH11 for the times shown (lanes 3–7). Cells were lysed, and Fos expression was monitored by immunoblotting (IB) with an anti-Fos antibody. b, human BJAB cells were transfected with the firefly luciferase reporter Fos-Luc3 or the mutant ΔF10H-Luc3, which lacks the SRE, and after culture for 24 h in low serum they were treated with CH11, ZVAD, or both, as indicated (DMSO, dimethyl sulfoxide vehicle). After 6 h, lysates were prepared and assayed as described. Luciferase activity is expressed as the ratio of relative light units (R.L.U.) and the β-galactosidase activity expressed from a co-transfected plasmid, whereby the value for untreated cells is set as unity. c, BJAB cells were transfected with the M20Fos-Luc reporter, which contains the M20 sequence in place of the SRE, and either control vector (pCMV5), MSRF, NMS4, or SRFC. After a 24-h culture in low serum, lysates were prepared and assayed for luciferase activity. Luciferase activity is expressed as the ratio of relative light units and the β-galactosidase activity expressed from a co-transfected plasmid, whereby the value for cells transfected with pCMV5 is set as unity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)It remained conceivable that the down-regulation of c-fosexpression could be due to something other than SRF cleavage. Since cleavage by caspases yields two SRF fragments, we chose to measure their direct influence on c-fos reporter expression. To this end, a mutant of SRF with altered DNA binding specificity was adopted. This mutant (MSRF), which was originally developed by Treisman and colleagues (38Hill C.S. Marais R. John S. Wynne J. Dalton S. Treisman R. Cell. 1993; 73: 395-406Abstract Full Text PDF PubMed Scopus (329) Google Scholar, 43Hill C.S. Wynne J. Treisman R. EMBO J. 1994; 13: 5421-5432Crossref PubMed Scopus (139) Google Scholar), contains part of the DNA-binding domain of MCM1 and binds to a sequence (M20) poorly recognized by SRF. A reporter containing the M20 element therefore has a low basal level of activity when transfected alone into BJAB cells (Fig. 5 c), which is elevated when MSRF is expressed in the cells. In contrast, expression of a carboxyl-terminal truncation of MSRF corresponding to the amino-terminal caspase fragment of SRF (NMS4) or the carboxyl-terminal fragment of SRF (SRFC) inhibits basal reporter expression slightly. Taken together, these results indicate that SRE-dependent c-fos expression is down-regulated early in apoptosis, that down-regulation is blocked by ZVAD, and that the fragments of SRF generated by caspase cleavage fail to maintain expression levels supported by full-length SRF.Given that the mutation of two predicted caspase sites produced a mutant of SRF resistant to caspase cleavage in vitro, we wished to see if the mutant was also resistant to cleavage in BJAB cells undergoing apoptosis. Thus, BJAB cells expressing various forms of SRF were treated with CH11, and SRF cleavage was assessed by immunoblotting. We observed that mutation of the site at Asp254 had little effect on SRF cleavage in vivo, whereas cleavage was severely impaired by mutation of the site at Asp245 (Fig. 6). As" @default.
• W2132038146 created "2016-06-24" @default.
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• W2132038146 date "2001-09-01" @default.
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• W2132038146 title "Serum Response Factor Cleavage by Caspases 3 and 7 Linked to Apoptosis in Human BJAB Cells" @default.
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