The Carboxyl-terminal Transactivation Domain of Human Serum ...

THE JOURNAL

OF

BIOLOGICAL CHEMISTRY

Voi. 268, No. 28, Iasue of October 5, pp. 21147-21154,1993 Printed in U.S.A.

The Carboxyl-terminal Transactivation Domain of Human Serum Response Factor Contains DNA-activated Protein Kinase Phosphorylation Sites* (Received for publication, February 23, 1993, and in revised form, May 17, 1993)

Shu-Hui LiuSO, Jing-Tyan Ma+, A. Yueh Yueh$, SusanP. L ~ - ~ i l l e r l l Carl l l , W. Andersonll**, and Sun-Yu NgS $$ From the $Institute of Molecular Bwbgy, Academia Sinica, NanKang, Taipei 11529, Taiwan, Republic of Chino, the §Institute of Life Science, National Tsing H w University, Hsinchu 30043, Taiwan, Republic of Chinn, and the llBwlogy Department, Brookhaven National Laboratory, Upton, New York 11973

The serum response factor (SRF)is a 67-kDa phos- activate the expression of downstream genes whoseproducts, phoprotein that, togetherwith auxiliary factors, mod- presumably, are required for DNA replication and cell diviulates transcription of immediate early genes contain- sion. ing serum responseelements in their promoters. Here The proto-oncogene c-fos has been an excellent paradigm we show that the carboxyl-terminal domain human of for the immediate early genes. The tran~riptionalactivation SRF is phosphorylated in vivo and is recognized in of the c-fos promoter by serum, growth factors, or other vitro by thedouble-strandedDNA-activated serine/ mitogenic signals depends on a 22-base pairs DNA sequence threonine-specific proteinkinase, DNA-PK. SRF phos- defined as the serum response element (SRE?’ (for review, phorylation by DNA-PKwas stimulated by its cognate binding site. Protein microsequence analysis of a 22- see Treisman (1992)).Serum response factor (SRF) is a 508amino acid synthetic SRF peptide andphospho~ptide amino acid, nuclear phosphoprotein that binds as a dimer to analysis of genetically altered glutathione S-transfer- the SRE (Prywes and Roeder, 1987;Treisman, 1987; Norman ase-SRF fusion proteins identified Ser-435 and Ser- et at., 1988). During SDS-polyacrylamidegel electrophoresis, 446 of human SRF as sites phosphorylated by DNA- the SRF polypeptide migrates with an apparent size of67 PK. Bothserines are followed by glutamine. Changing kDa; its abnormally slow electrophoretic mobility may be due to 0-glycosylation (Schroter et al., 1990; Reasonet al., 1992) Gln-436 and Gln-447 to otherresiduesreducedor eliminated p h ~ h o ~ l a t i oby n DNA-PK, confirming and/or to phosphorylation (Prywes et d.,1988, Manak et al., that these glutamines are important determinants for 1990; Misra et al., 1991). SRF produced by in vitro translation kinase recognition. The carboxyl-terminal transcrip- or purified from HeLa cells stimulates transcription in uitro, tion activation domain was mapped within a 71- amino suggesting that SRF functions as a positive transcription acid region that contains both DNA-PK phosphoryla- factor (Norman et al., 1988). Molecular cloningof the human tion sites. Amino acid substitutions that interferedwith SRF cDNA has facilitated the localization of its DNA-binding ph~pho~latio byn DNA-PKat Ser-435/446 inGAL4- and dimerization domain to a region within amino acids 133SRF fusion proteins were reduced in transactivation 264 (Norman et al., 1988). The carboxyl-terminal domain, potency. From these data we suggestthatDNA-PK from residue 265 to 508, also is well conserved through evophosphorylation may modulate SRF activity in vivo. lution (Mohun et al., 1991). We show here that this region has transactivation function in vivo. Protein phosphorylation/dephosphorylation is an important mechanism in mediating intracellular signal transducCellular proliferation is initiated by extracellular signals. tion. Many transcription factors are phosphorylated at disSerum stimulation of quiescent fibroblasts triggers the transtinct sites by more than one protein kinase (Hunter and mission of messages from cell surface receptors toward the Karin, 1992).SRF appears to undergo extensive modifications nucleus and causes cells to re-enter the cell cycle, leading to DNA replication and celldivision. The primary genomic following protein synthesis in serum-stimulated fibroblasts (Misra et al., 1991). Casein kinase I1 (CKII) phosphorylates targets of several signal transduction pathways are the immediate early genes (for review, see Lau and Nathans(1991)). SRF in vivo and in vitro (Manak et al., 1990; Marais et al., of CKII Many such genes encodetranscription factors, which, in turn, 1992;Janknecht et al., 1992). Although microinjection appears to stimulate c-fos transcription (Gauthier-Rouvikre * The work in the laboratory of S. N. is supported by Academia et al., 1991), CKII is already quite abundant in unstimulated Sinica and National Science Council (Republic of China). The costs cells, and SRF activation is not thought to be mediated of publication of this article were defrayed in partby the payment of directly through phosphorylation by CKII. Therefore, we inpage charges. This article must therefore be hereby marked “adver- itiated a search for other kinases that might modulate SRF tisement” in accordance with 18U.S.C. Section 1734 solelyto indicate activity. During this search we found that human SRF was this fact. I( Supported by a grant from the Exploratory &search Program of recognizedby a DNA-dependent serinelthreonine-specific the Brookhaven National Laboratory. Present address: Dept. of Bi- nuclear kinase, DNA-PK (Carter et al., 1990; Lees-Miller et oiogicai Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada. ** Supported in part by the Office of Health and Environmental &search of the United States Department of Energy. $4 To whom correspondence should be addressed. Tel.: 886-27899228; Fax: 886-2-7826085.

The abbreviations used are: SRE,serum response element; CAT, chloramphenicol acetyltransferase; CKII, casein kinase II; PCR, polymerase chain reaction; DNA-PK, DNA-activated protein kinase; GST, glutathione S-transferase; SRF, serum response factor; WGA, wheat germ agglutinin; PAGE, polyacrylamide gel electrophoresis.

21147

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Human SRF Phosphorylation by DNA-PK

ul., 1990; Lees-Miller and Anderson, 1991). We then mapped two DNA-PK phosphorylation sites to the carboxyl-terminal domain of SRF, which we showed is phosphorylated in vivo. This domain contains multiple glutamine and proline residues, a motif that occurs in the transcription activation domains of several transcription factors (Mitchell and Tjian, 1989). By testing various GAL4-SRF fusion genes, we then localized the carboxyl-terminal transcription activation domain of human SRF to a region that contains the DNA-PK phosphorylation sites. Finally, mutagenesis of the DNA-PK phosphorylation sites within this human SRF domain suggests that they may be functionally important in controlling SRF activity.

NaCl, 10 mM Tris-HC1 (pH 7.41, 1 mM EDTA) and lysed on ice in radioimmune precipitation buffer (50 mM Tris-HC1 (pH 7.4), 150 mM NaCL 0.5% sodium deoxycholate, 1%Nonidet P-40,0.1% SDS) with 1 mM phenylmethylsulfonyl fluoride, 2 pg/ml aprotinin, and leupeptin. SRF protein was immunoprecipitated with anti-SRF antibody, as described below, except that theradioimmune precipitation buffer was used in all washing steps. Purification of HeLa SRF-HeLa nuclear extracts were prepared as described (Dignam et al., 1983). HeLa SRF was purified by four chromatographic steps in the following order: DEAE-Bio-Gel Aagarose, Bio-Rex 70, double-stranded-DNA-cellulose, and wheat germ agglutinin (WGA)-agarose column chromatography. Purification of HeLa SRF by Bio-Rex 70 and double-stranded-DNA-cellulose chromatography was performed as described (Treisman, 1987). WGAagarose (Vector Laboratories, Burlingame, CA) affinity chromatography was performed as described (Jackson et al., 1990). Kinase Reaction and Immunoprecipitation-DNA-PK was purified MATERIALS ANDMETHODS through DEAE-Sepharose in the presence of 25 mM M&l2 as deCell Culture-NIH/STS fibroblasts (ATCC CRL 1658), HUT-12 scribed (Lees-Miller et al., 1990). The 350-kDa polypeptide accounts (Ng et al., 1989), and HeLa S3 (ATCC CCL 2.2) monolayer cultures for about 50-60% of total proteins; Ku autoantigen is presentin this were grown in Dulbecco’s modified Eagle’s medium (Life Technolo- preparation, gies, Inc.) supplemented with 10% fetal bovine serum. HeLa S3 Phosphorylation reactions were performed by adding 1pg of fusion suspension cultures were grown in RPMI 1640 medium (Life Tech- protein or about 0.1 pg of HeLa WGA-agarose-bound proteins to a nologies, Inc.) supplemented with 10% calf serum. 30-pl reaction mixture containing 10 pCiof [ Y - ~ ~ P ] A (5000 T P Ci/ Plasmid Constructions-To generate a plasmid encoding glutathi- mmol, Amersham Corp.), 10 mM MgCl,, 5 mM NaF, 2 pg/ml SRE one S-transferase-(GST) SRF fusion protein with the full-length oligonucleotide and 0.66 units of DNA-PK; the mixture was then human SRF, SRF cDNA (Norman et al., 1988) was amplified using incubated at 30 “C for 10 min. Labeled SRF was immunoprecipitated 5’-CCTGCATCGGGATCCATGTTACCGACCCAA-3’ asthe up- from the reaction mixture with 5 pl of affinity-purified polyclonal stream primer and 5’-TGTGTGTAAAGAATTCGTGAAAAGGC-3’SRF antibody on ice for 30 min, followed by a 1:l (v/v) slurry of as the downstream primer. The PCR product was inserted in frame protein A-agarose in dilution buffer (10 mM Tris-HC1 (pH 8.0), 0.14 into thepGEX-2T polylinker (Smith andJohnson, 1988).To generate M NaCl, 0.1% Triton X-100, 1%bovine hemoglobin, 0.025% NaN3), a plasmid encoding the GST-SRF fusion protein with human SRF and then themixture was rotated at 4 “C for 90 min. The protein Aamino acids 266-508, the above pGST-SRF-(1-508) was first digested agarose was pelleted and washed sequentially twice with dilution with BamHI and NaeI, blunt-ended, and then self-ligated. buffer, once with TSA buffer (10 mM Tris-HC1 (pH 8.0), 0.14 M NaCl, All GAL4-SRF fusion genes were constructed with the pSG424 0.025% NaN3), and once with 0.05 M Tris-HC1 (pH 6.8). vector (Sadowski and Ptashne, 1989). GS-3 was constructed by amPhosphopeptide Mapping and Two-dimensional Phosphoamino plifying the SRF cDNA segment encoding Asp-261 to Glu-508 by Acid Analysts-Phosphopeptide analysis by two-dimensional separaPCR and cloning this fragment into theBamHI and XbaI sites of the tion on thin-layer cellulose plates was carried out as described (Boyle pSG424 vector. To construct GS-4, GS-3 DNA was digested with et al., 1991) with a Hunter thin-layer peptide mapping electrophoresis BamHI and PpuMI, blunt-ended, and then self-ligated. GS-6 was system (C. B. S., Del Mar, CA). Immobilized 32P-labeledpolypeptides constructed by self-ligation after SmaI and MscI digestion of GS-3 were digested in 200 pl of NH4HC03containing 10 pg ofchymotrypsin DNA. To construct GS-7, GS-3 DNA was digested with AatII and for 2 h at 37 “C, and then another 10 pg of fresh enzyme was added PflMI, blunt-ended, and then self-ligated. To construct GS-8, GS-6 for overnight incubation. The dried samples were dissolved in pH 1.9 DNA was digested with PflMI and XbaI, the recessed 3’ termini were buffer then subjected to first dimension TLC electrophoresis at 1.25 then filled with Escherichia coli DNA polymerase I Klenow fragment, kV for 35 min. Ascending chromatography in the second dimension after which the protruding termini were removed with mung bean was performed in n-butano1:acetic acidpyridine:HzO (75:15:5060, nuclease, and the blunt-ended plasmid was self-ligated. To generate v/v). Phosphopeptide mapping on SDS-PAGE was performed as dea plasmid encoding the GST-SRF fusion protein with human SRF scribed (Flannery et al., 1989). Phosphoamino acid analysis by twoamino acids 406-508, GS-6 DNA was digested with HincII and XbaI for cloning into the SmaI and XbaI sites of the pGEX-KG vector dimensional separation on thin-layer cellulose plates was carried out as described (Boyle et al., 1991). (Guan and Dixon, 1991). Transfections and CAT Assays-HUT-12 cells were transfected by Plasmids that express serine or glutamine mutants were constructed by amplifying the DNA segment encoding Asn-432 to Glu- calcium phosphate-mediated precipitation, with a total of 10 pg of 508 with PCR primers containing altered codons and cloning such DNA (4 pg of GAL4-SRF effector plasmid or pSG424 vector plasmid, fragments into the BsmI sites of the GS-3 plasmid. GS-3 mutants 4 pgof G5BCAT reporter plasmid, and 2 pgof pCMVp internal were digested with SmaI and MscI, then self-ligated to yield the control plasmid) added per 60-mm dish. Cells were incubated with corresponding GS-6 mutants. To generate plasmids encoding mutant precipitates for 4h, kept in Dulbecco’smodifiedEagle’smedium GST-SRF-(266-508) fusion proteins, the appropriate GAL4-SRF- supplemented with 0.5% fetal bovine serum, and harvested 36 h later. Cell extracts were prepared, and CAT assays were performed as (261-508) plasmid was digested with PpuMI and HpaI, andthe resulting 597-base pairs PpuMI-HpaI restriction fragment containing previously described (Gorman et al., 1982). p-galactosidase activity fluoromethese changes was inserted into thepGST-SRF-(266-508) expression was determined by 4-methyl-umbelliferyl-~-~-galactoside plasmid in place of the wild-type fragment. DNA sequencing of the tric assay as described (MacGregor et al., 1989) to normalize CAT above GST-SRF and GAL4-SRF fusion genes confirmed the fidelity activity. To check the expression of each GAM-SRF Construct 36 h after transfection, cells were labeled in 0.5 mCi/ml ~ - [ ~ ~ S l m e t h i o n i n e of PCR reactions and that thefusions were in-frame. Expression of GST-SRF Fusion Proteins-The expression and for 1 h. Immunoprecipitations were then performed as described purification of GST fusion proteins were carried out as described above. (Smithand Corcoran, 1990). TheGST portion wasremoved by thrombin cleavage. RESULTS Preparation and Affinity Purification of Anti-SRF AntibodiesPhosphorylation of SRF in Vitro-SRF phosphorylation Purified GST-SRF fusion protein expressed from pGST-SRF-(266508) was used to produce polyclonal antisera from female rabbits. probably occurs primarily in the nucleus (Misra et al., 1991); Antibodies that recognize the GSTportion of the fusion protein were therefore, we first fractionated nuclear extracts for kinases removed by passing the serum over a column containing purified that might phosphorylate SRF. A SRF kinase and apreviously GST coupled to Affi-Gel 10 beads (Bio-Rad). characterized DNA-activated protein kinase, DNA-PK I n Viuo 3zPLabeling-HeLa S3 monolayer cultures, about 70% confluent, were labeled with 1.3 mCi/ml [32P]orthophosphate(Amer- (Carter et al., 1990; Lees-Miller et ul., 1990),copurified (results sham Corp.) for 2 h. After labeling, the medium was removed, and not shown). Fig. 1 shows that SRFphosphorylation by DNAthe cultures were washed twice with ice-cold STE buffer (150 mM PK was stimulated both by the SRE element (lane 2) and an

Human SRF Phosphorylation by DNA-PK 1

2

3

21149

4

carboxyl-terminal SRF fragment (residues 266-508) as glutathione S-transferase-SRF fusion proteins. Both the fulllength SRF and the SRF carboxyl-terminal fragment were 200efficiently phosphorylated by DNA-PK the GST fragment alone was not phosphorylated by DNA-PK (data not shown 97 and Fig. 3, lanes 1 and 2). The DNA-PK phosphorylation sites in the SRF carboxyl6s+p67SRF terminal regionwere further localized by two-dimensional phosphopeptide mapping of chymotrypsin digestion products 43(Fig. 2). One of the major DNA-PK-labeled chymotryptic phosphopeptides was spot C2a (panel C). Human SRF labeled in uiuo with 32Palso yielded spot C2a (panels A and B ) . The identity of the spots from in vitro and in uiuo labeled SRF 29 preparations was confirmed by mixing (panel D).Phosphopeptide C2a accounted for about 4% of the total SRF radioactivity from serum-starved Hela cells (panel A ) and 5% of FIG. 1. Phosphorylation of human SRF by DNA-PK is total radioactivity in SRF from serum-stimulated HeLa cells DNA-dependent. WGA-agarose-purifiedHeLa SRF was incubated (panel B ) . Therefore, phosphorylation sites within this pepwith DNA-cellulose purified DNA-PK and [y3*P]ATPin the presby serum-inducible kinase ence of the SRE oligonucleotide ( l a n e 2), the AP-1 oligonucleotide tide apparently are not targeted ( l a n e 3), or without added DNA ( l a n e I ) ; after incubation with antiSRF antiserum, the immunoprecipitates were analyzed by SDSPAGE. DNA-PK was not added to a control reaction ( l a n e 4 ) . The sizes of marker proteins, in kilodaltons, are indicated. The position of the 67-kDa SRF is indicated on the autoradiogram.

AP-1 oligonucleotide (lane 3) in comparison with phosphorylation in the absence of added DNA (lane 1). SRF phosphorylation also was stimulated by poly(d1-dC).poly(dI-dC) (data not shown). When DNA-PK was omitted from the reaction, SRF was not labeled (lane 4 ) . Thus, under the in vitro conditions used here, phosphorylation of human SRF by DNA-PK does not require the recognition of specific DNA sequences by either SRF or DNA-PK. DNA-PK has been shown to be activated by a wide variety of linear, doublestranded DNAs and oligonucleotides (Carter etal., 1990;LeesMiller et al., 1990);phosphorylation also may be enhancedby the simultaneous binding of kinase and substrate to the same DNA fragment (Lees-Miller and Anderson, 1991; Anderson and Lees-Miller, 1992; Gottlieb and Jackson, 1993). Mapping of in Vivo and the DNA-PK Phosphorylation Sites-Based on an in uitro transcription assay, the transactivation domain of human SRF was localized to thecarboxylterminal domain (Prywes and Zhu, 1992). Phosphorylation of this domain has notbeen reported previously. Endoproteinase Glu-C digestion of human SRF from 32P-labeled cells produced a major phosphopeptide with the mobility of a 29-kDa peptide (data notshown). Phosphopeptides with lower apparent molecular weights, from about 15 to 1.5 kDa, also were observed. A similar pattern of phosphopeptides was obtained from in uiuo labeled mouse SRF after endoproteinase Glu-C digestion, except that a 1.5-kDa peptide was not detected. Two-dimensional phosphoamino acid analysis of the 29-kDa human peptide revealed only phosphoserine, as previously reported for intact SRF(Prywes et al., 1988). Endoproteinase Glu-C cleaves polypeptides specifically after glutamic acid residues; only two such residues, Glu-450 and Glu-493, are found in the carboxyl-terminal half. Proteolytic cleavage a t the Glu-450-Pro-451 peptide bond is presumed to be inefficient (Drapeau, 1977). Therefore, only the SRF carboxylterminal domain is devoid of endoproteinase Glu-C sites over a sufficiently long stretch (235 residues) to account for a 29kDa Glu-C peptide. We surmise that in vivo human SRF is phosphorylated at serine residues located in the carboxylterminal domain between residues Thr-259 and Glu-493. To determine if DNA-PK phosphorylates the carboxylterminal domain of SRF, we constructed plasmids that expressinbacteria full-length SRF (residues 1-508) and a

serum-stimulated serum-starved -

+

Electrophoresis d

in vitro

+

Electrophoresis

-

mixing

-

FIG. 2. Two-dimensional phosphopeptide mapping of in vivo labeled SRF and in vitro phosphorylated SRF carboxyl-

terminal domain. Panel A , immunoprecipitates of SRF from serumstarved HeLa S3 cells labeled with [32P]orthophosphatein the presence of 100 nM okadaic acid were analyzed by 10% SDS-PAGE and visualized by autoradiography. The SRF band was excised and digested with chymotrypsin, and thedigestion products were applied to thin-layer cellulose plates and resolved in the horizontal dimension by electrophoresis at pH 1.9 (anode to the left) and in the vertical dimension by ascending chromatography as described (Boyle et al., 1991). The plates were exposed to x-ray film for 4 days at -70 "C using an intensifying screen. The origins (ori) of migration are indicated. Panel B, immunoprecipitates of SRF from serum-stimulated HeLa S3 cells, labeled with [32P]orthophosphateas above and processed for two-dimensional chymotryptic peptide mapping as described in panel A. Panel C, affinity-purified GST-SRF-(266-508) fusion protein was digested with thrombin and repurified to remove the GST fragment. The SRF carboxyl-terminal fragment was incubated with DNA-PK, [Y-~*P]ATP, andSRE oligonucleotide, and reactions were analyzed by SDS-PAGE. The 30-kDa band, visualized by autoradiography, was excised and processed for two-dimensional chymotryptic peptide mapping as described in panel A. Panel D, equal amounts of phosphopeptides from panels B and C were processed as described above.

Human SRF Phosphorylation by DNA-PK

21150

cascade(s). Another spot,labeled as C2b, also contained DNAPK phosphorylation site(s) (panel C) and was phosphorylated in uiuo (panelsA and B ) . The phosphopeptide(s) derived from CKII phosphorylation of a fusion protein containingthe NH2terminal sites did not migrate during chromatography and appeared as a horizontal streak (data not shown). Similar streaks and phosphopeptide C1 were observed in samples labeled in uiuo (panels A and B ) but not in samples labeled in uitro with DNA-PK (panel C ) . Identification of SRF Residues Phosphorylated by DNAPK-DNA-PK recognizes serines in SV40 T-antigenand threonines in HSP9Oa that are followed immediately by glutamine (Lees-Miller and Anderson, 1991) and peptides from the p53 tumor suppressor protein that contain-SQ-sequences (Lees-Miller et al., 1992). Other known DNA-PK substrates including human Spl (Jackson et al., 1990), Oct-1, TFIID, andc-jun (Lees-Miller and Anderson, 1991) also contain multiple SIT-Q motifs. These findings suggested that probable DNA-PK phosphorylation sites in the SRF carboxylterminal domain are Ser-435 and Ser-446, the only two serines in SRF followed immediately by glutamine. Totestthis hypothesis we synthesized a peptide corresponding to the human SRFsequence from Val-430 to Pro-451. This peptide was phosphorylated by DNA-PK in a DNA-dependent manner, and, as expected, the amino acids phosphorylated were serines (data not shown). To determine the specific serine residue(s) phosphorylated, we converted the peptidyl phosphoserine produced by DNA-PK to S-ethylcysteine; then, the sequence of the modified peptide was determined by automated Edman degradation (Chen et al., 1991). Phenylthiohydantoin-S-ethylcysteine was detectedin two sequencer cycles, cycle 6, corresponding to Ser-435, and cycle 17, corresponding to Ser-446, but not inother cycles (data notshown). This result indicated that Ser-435 and Ser-446 are preferred DNA-PK targets. To testwhether the adjacent Gln residues are essential for DNA-PK recognition, we tried to phosphorylatea second peptide that was identical except that glutamine residues Gln436 and Gln-447 were changed to glutamic acids; this mutant peptide was no longer phosphorylated (data not shown). We also expressed a Glu-4361447 mutant protein in E. coli and

compared its phosphorylation with the wild-type protein (Fig. 3). The 30-kDa carboxyl-terminal fragment from the thrombin cleaved mutant GST-SRF (aa 266-508) protein was still phosphorylated by DNA-PK (lane 3) but at a 3- to 4-fold lower rate than the wild-type fragment (lane 1). We then measured the ratio of phosphoserine to phosphothreonine in the wild-type and Glu-4361447mutant SRFcarboxyl-terminal fragment (Fig. 3C). This ratio was about 4 for the wild-type fragment, but the mutant fragment had nearly equal amounts of phosphoserine and phosphothreonine, indicating that phosphorylation at one or more serine sites, presumably Ser-435 and/or Ser-446, was much reduced by elimination of the two glutamines. We have not identified the threonine(s) phosphorylated by DNA-PK, but Thr-380 is a likely site because this residue is followed by Gln-381. At least one additional DNA-PK site is present in the Asp-261 to Gly-405region since spot C3 was missing from a GST-SRF fusion protein truncated from Ala-266 to Gly-405 (Fig. 4, panel D). Glu-C cleaved the wild-type SRF carboxyl-terminal fragment into phosphopeptides with apparent sizes of 24 and 8 kDa (Fig. 3B,lane 4 ) . We presume these bands correspond to Ala-266-Glu-493 and Gly-426-Glu-493, respectively. The identity of the amino terminusof the 24-kDa polypeptide was confirmed by protein sequence analysis. Cleavage of Asp-Gly bonds by endoproteinase Glu-C is known to occur in other substrates under digestion conditions that areotherwise specific for cleavage after glutamic acid (Drapeau, 1977). After cleavage of the mutant fragment, only one 20-kDa phosphopeptide was observed (Fig. 3, lane 5); the 8-kDa Glu-C fragment (Gly-426-Glu-493)was no longer detected. The smaller size of the large fragment and elimination of the 8-kDa fragment were expected from the introduction of two potential Glu-C cleavage sites after residues 436 and 447. These results further confirm the location of Ser-435 and Ser-446 within. the 8-kDa fragment. To verify that phosphopeptide C2a and/or C2b correspond to the Ser-435-Phe-458 chymotryptic peptide containing the Ser-4351446 sites, we subjected the Glu-4361447 mutant and an Ala-4351446 mutant to two-dimensional phosphopeptide analysis after phosphorylation with DNA-PK (Fig. 4). Both phosphopeptides were missing from the mutants (panels B

C

B

A

Glu-C phosphopeptides

wt us mutant

1

u-

2

4

3

'

~

n~-3OkDa

n1I-

5 I

"

II-

'"

. )

Phosphonmino acids

.-

-

435 446

FIG. 7. Human SRF domains and phosphorylation sites. The three functional domains of human SRF are indicated within the rectangle representing the 508-amino acid SRF polypeptide. The centraldomain, between residues 133 and 222, is essential for DNA binding and dimer formation (Norman et al., 1988). The carboxyl-terminal domain functions in transcriptional activation. The 29-kDa carboxylterminal phosphopeptide produced by endoproteinase Glu-C digestion of SRF labeled in vivoafter serum stimulation of serum-starved HeLa S3 cultures probably contains residues 259-450. Two-dimensional phosphopeptide mapping of in vivo labeled SRF digested with chymotrypsin identified phosphopeptides C2a/C2b, Ser-435-Phe-458 as one of the major in vivo phosphopeptides (see Fig. 2, panels A and B ) . Twodimensional phosphopeptide mapping of the in vitro phosphorylated carboxyl-terminal peptide digested with chymotrypsin identified the same phosphopeptides C2a/C2b, Ser-435-Phe-458 as themajor DNA-PK phosphopeptides (see Fig. 2, panel C). A syntheticpeptide, Val-430Pro-451, was used to identify Ser-435 and Ser-446 as sites of DNA-PK phosphorylation. Also shown is the sequence around previously identified CKII phosphorylation sites a t Ser-77, Ser-79, Ser-83, and Ser-85 (Manak and Prywes, 1991; Marais et al., 1992; Janknecht et al., 1992).

at Ser-4351446. Wealso show that the ability of DNA-PK to recognize Ser-435 and Ser-446, both in a synthetic peptide and in bacterial expressed protein, is dependent upon the presence of the adjacent glutamine residues, Gln-436 and Gln447, thus confirming the importance of these residues in site recognition by DNA-PK. Our two-dimensional phosphopeptide maps showed that the Ser-4351446 containing phosphopeptides C2a/C2b were phosphorylated in uiuo. Although we cannot rule out possible phosphorylation of other serines in this region by anotherprotein kinase in uiuo, ourresults strongly suggest that Ser-435 and Ser-446 may bephosphorylated in uiuo. Both SRF and DNA-PK are moderately abundant nuclear proteins; thus, it is reasonable to suppose that DNA-PK would have access to SRF inuiuo. Regulation of SRF Activity by Phosphorylation-Covalent modification of human SRF may serve several functions. Phosphorylation by CKII of the Ser-77-Ser-85 site increases the rate of SRF-binding site exchange (Marais et al., 1992; Janknecht et al., 1992). This modification may be important to permit a rapid exchange of factors at SREs. Phosphorylation may also serve to regulate the interaction of SRF with other factors. SRF and ~ 6 2 form ~ ' ~a ternary complex with the SRE, and thisinteraction seems to be important for the serum inducibility of the c-fos promoter in vivo (Shaw et al., 1989; Hill et al., 1993) and may be regulated by ternary complex factor phosphorylations (Graham and Gilman, 1991; Gille et al., 1992; Marais et al., 1993). No effect of CKII phosphorylation, or mutation of the amino-terminal CKII sites, on the ability of ~ 6 2 to ~ form ' ~ a ternary complex with SRF and the SREwas observed (Janknecht et al.,1992; Gille et al., 1992). Finally, phosphorylation could affect the interaction of SRF with other components of the transcription complex, such as the Tax1protein of human T-cell lymphotrophic virus-1 (Fujiiet al., 1992). Two of the major DNA-PK phosphorylation sites arein the carboxyl-terminal segment of SRF, between His-406 and His476, that is responsible for transcriptional activation. Since

Ser-435 and 446 likely are phosphorylated in uiuo, a direct role for DNA-PK phosphorylation in the modulation of human SRF transcriptional activity is a possibility. Our data from the analysis of mutant GAL4-SRF fusion proteins changed at the Ser-4351446 sites and the Gln-436/447 sites areconsistent with such a role. Changing Ser-4351446 to alanine decreased transcriptional activation in HUT-12 cells to an average of 40%. In contrast, changing Gln-4361447 to glutamic acid increased expression of the GAL4 sites-dependent reporterabout 3-fold, while changing Gln-4361447 to lysine had little effect on activity. Thus, Ser-435/446 are not critical for transcriptional activation in this artificial situation; however, the decreased activity of the alanine-substituted constructs and the increased activity of the glutamic acid-substitutedconstructsareconsistent with a possible enhancement of transactivation ability by phosphorylation of Ser-4351446. The relatively small decrease in activity displayed by the alanine-substitutedconstructs is not unexpected, because only a fraction of the expressed protein may be phosphorylated in uiuo. Furthermore, modifications that alter a rate-limiting step in nature may not have as great an effect when protein fragments are overexpressed in an artificial environment. Clearly, additionalmutantsandfurther analysis will be required to determine if enhanced transcriptional activity resulted from an increased negative charge density (Mitchell and Tjian, 1989) or from other structural effects (Regier et al.,1993). In addition to SRF, DNA-PKphosphorylates several other transcription factors (see Lees-Miller and Anderson (1991) and Anderson and Lees-Miller (1992)); however, the precise role of DNA-PK in transcription control is still very much unclear. If DNA-PK behaves as aconstitutively active protein kinase that is continuously present on chromatin, then its function may beto induce a structuralchange in transcription factors when they bind to their recognition elements. In this case it would be unlikely that DNA-PK plays a direct role in serum-stimulated activation of transcription. Alternatively,

21154

Human SRF Phosphorylation by DNA-PK

DNA-PK may be activated only under special circumstances. The discovery that theKu autoantigen, a major cellularDNA end-binding protein (de Vries et al., 1989; Blier et al., 1993), is a kinase cofactor (Dvir et al., 1992; Gottlieb and Jackson, 1993) and that kinase activation in vitro requires DNA ends (Carter et al., 1990; Anderson and Lees-Miller, 1992; Gottlieb and Jackson, 1993) is suggestive of a role for DNA-PK in response to DNA damage. A possible invohement of SRF in UV-induced c-fos activation has been proposed (Stein et al., 1989). c-Jun and p53, two other transcription factors phosphorylated by DNA-PK (~es- iller et ul., 1992; Wang and Eckhart, 1992; Bannister et al., 1993), also may be involved in mediating cellular responsesto DNA damage (Stein et al., 1989; Lane, 1992). These observations raise the intriguing possibility that DNA-PK maybe a component of a DNA damage-detectionsystem and may play a roleincoupling transcription and DNA repair (Schaeffer et al., 1993). The identi~cationof SRF as a substrate for DNA-PK and elucidation of the target sites for phosphorylation should facilitate future studies on the in uiuo functions of the DNA-PK. Ku autoantigen complex.

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