Cell, Vol. 64, 573-584, February8, 1991,Copyright © 1991 by Cell Press

Activation of Protein Kinase C Decreases Phosphorylation of c-Jun at Sites That Negatively Regulate Its DNA-Binding Activity William J. Boyle,*t Tod Smeal,t Libert H. K. Defize,§ Peter Angel,§ II James R. Woodgett,# Michael Karin,§ and Tony Hunter* * Molecular Biology and Virology Laboratory The Salk Institute La Jolla, California 92037 Department of Biology § Department of Pharmacology University of California at San Diego La Jolla, California 92093 #Ludwig Institute for Cancer Research 91 Riding House Street London WIP 8BT England

Summary In resting human epithelial and fibroblastic cells, c-Jun is phosphorylated on serine and threonine at five sites, three of which are phosphorylated in vitro by glycogen synthase kinase 3 (GSK-3). These three sites are nested within a single tryptic peptide located just upstream of the basic region of the c-Jun DNAbinding domain (residues 227-252). Activation of protein kinase C results in rapid, site-specific dephosphorylation of c-Jun st one or more of these three sites and is coincident with increased AP-l-binding activity. Phosphorylation of recombinant human c-Jun proteins in vitro by GSK-3 decreases their DNAbinding activity. Mutation of serine 243 to phenylalanine blocks phosphorylation of all three sites in vivo and increases the inherent trans.activation ability of c-Jun at least lO-fold. We propose that c-Jun is present in resting cells in a latent, phosphorylated form that can be activated by site-specific dephosphorylation in response to protein kinase C activation. Introduction The phorbol ester tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) induces a variety of changes in cell metabolism, morphology, and growth by directly affecting the activity of protein kinase C (PKC) (Nishizuka, 1986). Among these processes are the immediate transcriptional activation of viral and cellular genes, such as those encoding the SV40 T antigens, collagenase, metalIothionein IIA, and stromelysin (Imbra and Karin, 1986, 1987; Angel et al., 1987a, 1987b; Lee et al., 1987; Kerr et al., 1988). Analysis of the promoter regions of these genes 1Present address: Department of Molecular and Cellular Biology, AMGEN Center, 1900 Oak Terrace Lane, ThousandOaks, California 91320. IIPresentaddress: Institutfur Genetik und Toxikologie,Kernforschungszentrum Karlsruhe,D-7500Karlsruhe,FederalRepublicof Germany.

led to the identification of the cis-acting TPA response element (TRE) (Angel et al., 1987b; Lee et al., 1987). The consensus TRE, 5'-TGAC/GTCA-3', is recognized by transcription factor AP-1 (Angel et al., 1987b; Lee et al., 1987). In addition to TPA, various growth factors and cytokines can also activate PKC and subsequently induce AP-l-responsive gene expression (reviewed by Karin, 1990). AP-1 is a complex consisting of several polypeptides (Angel et al., 1987b; Bohmann et al., 1987; Lee et al., 1987). A major component of AP-1 is a protein encoded by the normal cellular homolog of the avian v-jun oncogene, c-jun (Bohmann et al., 1987; Angel et al., 1988a). The c-Jun protein is a transcription factor that is both necessary and sufficient for the transcriptional activation of AP-l-responsive genes (Angel et al., 1988a, 1988b; Chiu et al., 1988; Bohmann and Tjian, 1989; Abate et al., 1990a). Bacterially expressed c-Jun protein binds specifically to various TREs as a preformed dimer (Angel et al., 1988a; Smeal et al., 1989; Abate et al., 1990b). Mutational analysis of the TRE indicates that c-Jun/AP.1 binding to the TRE is essential for TPA inducibility (Angel et al., 1987b, 1988a, 1988b; Chiu et al., 1988). An additional component of AP-1 is the rapidly inducible product of the c-los proto-oncogene, c-Fos (Rauscher et al., 1988a; Chiu et al., 1988; Sassone-Corsi et al., 1988a). c-Jun and c-Fos form a stable heterodimer via a coiled-coil interaction of a region known as the leucine zipper (Kouzarides and Ziff, 1988; Landschulz et al., 1988; SassoneCorsi et al., 1988b; Gentz et al., 1989; Schuermann et al., 1989; Smeal et al., 1989; Turner and Tjian, 1989). Although c-Jun and other Jun-related proteins form thermostable homodimers, c-Fos does not homodimerize and only forms stable heterodimers with the members of the Jun family (Nakabeppu et al., 1988; Halazonetis et al., 1988; Zerial et al., 1989). The stability of the c-Jun-c-Fos heterodimer is significantly greater than that of the c-Jun homodimer (Rauscher et al., 1988b), although both complexes appear to have equal affinity for the TRE (Smeal et al., 1989). Transient expression of c-Jun in cells with low endogenous AP-1 activity, such as undifferentiated F9 cells, results in the formation of homodimers capable of activating TRE-containing reporter genes (Chiu et al., 1988; Sassone-Corsi et al., 1988a; Smeal et al., 1989). Coexpression of c-Jun and c-Fos results in more efficient activation of AP-l-responsive genes because of the increased stability of the heterodimer (Chiu et al., 1988; Rauscher et al., 1988b; Smeal et al., 1989). Thus, one would predict that cellular factors that influence the ability of c-Jun to dimerize with itself and c-Fos and/or that modulate dimer affinity for the TRE are crucial determinants involved in modulation of AP-1 activity. The mechanism by which TPA activates AP-1 is not well understood, but since activation of certain AP-l-responsive genes and an increase in AP-1 activity can occur in the absence of new protein synthesis (Angel et al., 1987b, 1988b; Chiu et al., 1987), it is generally believed to involve

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Figure 1. Analysis of c-Jun Phosphorylation during TPA-Induced Activation of AP-1 (A) Anti-Jun immunoprecipitates (above) were made from HeLa TK- cells metabolically labeled with either Tran3SS-labelor [32p]orthophosphate, and then treated with 100 ng/ml TPA for 30 min (+) or left untreated as a control (-) as described in Experimental Procedures. The immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis on a single gel. The 35S lanes were fluorographed and exposed for 12 hr; the 32P lanes were autoradiographedand exposed for 8 hr. Partial amino acid hydrolysates (below) of 32p-labeled c-Jun purified as detailed above were separated in two dimensions as described in Experimental Procedures, and 32P-labeled phosphoamino acids were detected by exposure to presensitized film and an intensifying screen for 5 days at -70°C. The indicated phosphoamino acid standards were located by ninhydrin staining. (B) Serum-starved human osteosarcoma MG63 and HeLa TK~ cells were labeled with [ 32P]orthophosphats, then treated with TPA (100 ng/ml) for 30 min or left untreated, c-Jun was purified by immunoprecipitation and SDS-polyacrylamide gel electrophoresis, and subjected to tryptic digestion as described in Experimental Procedures. Tryptic digests (,'.,250 cpm of each) were applied to thin-layer cellulose plates, then resolved in the horizontal dimension by electrophoresis at pH 1.9 (anode to the left) and the vertical dimension by ascending chromatography as described in Experimental Procedures. Phosphopeptides were detected by exposure to presensitized film and an intensifying screen for 3 days at -70°C. Peptides identified as a, b~ and b2, and c are described in the text. (a) Human osteosarcoma MG63 cells. (b) HeLa TK- cells. (c) Schematic diagram of c-Jun phosphotryptic peptides. The phosphoamino acid content of the lettered peptides is indicated. The origin, which is indicated by a vertical arrowhead, is located in the position where samples were applied for (a) and (b).

posttranslational modification(s) of preexisting AP-1. Since TPA directly activates PKC (Nishizuka, 1986), it is likely that p h o s p h o r y l a t i o n is a crucial modification for up-regulating AP-1 activity. Consistent with this notion, both c-Fos and c-Fos-associated protein p39/c-Jun are known to be phosphorylated in vivo (Curran et al., 1984; Barber and Verma, 1987; M011er et al., 1987; Franza et al., 1988). To investigate the role of p h o s p h o r y l a t i o n in modulating AP-1 activity, w e have analyzed the phosphorylation of c-Jun during the AP-1 induction response. Our results suggest that resting cells contain a latent form of phosphorylated c-Jun incapable of efficient binding to the TRE. TPA stim-

ulation results in the rapid d e p h o s p h o r y l a t i o n of c-Jun at specific sites and activation of its DNA-binding function. Results c-Jun Is e N u c l e a r P h o s p h o p r o t e i n To determine w h e t h e r c-Jun is a phosphoprotein and whether TPA treatment affects its phosphorylation, we isolated c-Jun from ~ S - and 32p-labeled HeLa cells using an anti-trpE-c-Jun serum (Chiu et al;, 1988). Although this antibody may recognize other Jun family members, unstimulated HeLa cells or cells treated for less than 60 min

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Figure 2. Time Course of TPA-Induced Dephosphorylation of c-Jun in Quiescent Normal Human Diploid Fibroblasts Flow 2000 human embryonic lung fibroblast cultures were subpassaged into 6 cm culture dishes, grown to 90% confluence in DMEM containing 10% FBS, then shifted to DMEM containing 0.5% FBS for 48 hr prior to labeling with [32P]orthophosphate for 2.5 hr. Cultures were treated with either 100 ng/ml TPA or 20 pM forskolin for the indicated times, c-Jun was purified and subjected to tryptic mapping as described in Experimental Procedures and the legend to Figure 1. The amounts of radioactivity analyzed were 0 min TPA, ,',80 cpm; 15 min TPA, ~,120 cpm; 30 min TPA, ,'~130 cpm; 60 min TPA, ,'~125 cpm; 60 rain forskolin, "~65 cpm. Shown are autoradiograms of a 10 day exposure to presensitized film at -70°C using an intensifying screen.

with TPA express primarily c-jun mRNA (Chiu et al., 1989) and protein (J. Meek and M. Karin, unpublished data). A single 40 kd c-Jun protein was precipitated from 35Slabeled cells (Figure 1A). As previously reported, no increase in newly synthesized c-Jun was detected after 30 min of TPA treatment (Angel et al., 1988b). A single protein comigrating with c-Jun was isolated from 32p-labeled lysates (Figure 1A), which contained both phosphoserine (RSer) and phosphothreonine (RThr) (but not P3"yr)at a ratio of about 5:1 (Figure 1A). This protein was not immunoprecipitated by control preimmune serum, and its precipitation was blocked by pretreating the anti-c-Jun antibodies with ,~1 p.g of unlabeled cognate antigen (data not shown). No increase in 3~p-labeled c-Jun was observed within 30 min of TPA treatment (Figure 1A). c-Jun isolated with antic-Fos antibodies from TPA-induced cells as part of the c-Jun-c-Fos complex also contained RSer and RThr (data not shown). Phosphorylated c-Jun has been detected in a wide variety of cell lines of various lineages (fibroblastic, epithelial, hematopoietic) from avian, murine, and human sources (W. J. Boyle and G. Glenn, unpublished data). TPA Induces Site-Specific Dephosphorylation of c-Jun Protein Although TPA did not induce obvious changes in total c-Jun phosphorylation, we determined whether changes in the phosphorylation of individual sites had occurred by two-dimensional phosphopeptide mapping of c-Jun from 32p-labeled human osteosarcoma (MG63) and epithelial (HeLa) cells, c-Jun from serum-starved MG63 cells contained three major and one minor phosphopeptide (Figure 1B, [a]). Peptide a contained P.Ser and P.Thr, indicating that it was phosphorylated on more than one site, and peptide b contained only RSer. Suprisingly, TPA treatment of MG63 cells resulted in the complete disappearance of peptide a, but not of other peptides (Figure 1B, [a]), indicating net dephosphorylation of a site(s) in this peptide.

c-Jun from HeLa cells gave similar maps (Figure 1B, [b]), although we detected two additional peptides, peptide b2, which contained equal amounts of P.Ser and P3"hr, and peptide c, which contained only P.Ser(Figure 1B, [c]). Peptide b2 was only detected in c-Jun isolated from HeLa cells and may represent a different phosphorylation isomer of this peptide (vide infra). TPA treatment of HeLa cells also resulted in the disappearance of peptide a. No overt changes in phosphopeptides x and y were observed during these treatments. To extend these results, we analyzed the phosphorylation state of c-Jun in quiescent normal human diploid fibroblasts, Flow 2000, after varying times of TPA treatment by peptide mapping. In c-Jun from quiescent Flow 2000, peptides a and b were present, but we also detected a minor peptide (z) not present in c-Jun from either HeLa or MG63 cells (Figure 2, leftmost panel). TPA treatment led to a substantial decrease in the relative amount of peptide a within 15 min, and the complete disappearance of peptide a and an increase in the relative amount of peptide b within 30 min. These changes were no~tobserved in cells treated for 60 min with forskolin, an indirect activator of cAMP-dependent protein kinass (PKA), which does not induce AP-1 activity (Chiu et al., 1989) (Figure 2, rightmost panel). Phosphorylation of the c-Jun DNA-Binding Domain In Vitro with GSK-3 To investigate the role of protein phosphorylation in regulating c-Jun activity, we searched for known protein kinases that could phosphorylate c-Jun on physiologically relevant sites. Although AP-1 activity is stimulated by TPA, neither PKC nor PKA phosphorylated c-Jun or other AP-1 components in vitro (W. J. Boyle and E. Allegretto, unpublished data; Hal et al., 1988). We then tested glycogen synthase kinase 3 (GSK-3), because we have recently identified it as a Myb protein kinase that phosphorylates a

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Figure 3. GSK-3 Phosphorylation of c-Jun (A) Alignment of the C-terminal sequence of glycogen synthase phosphorylated by GSK-3 (and casein kinase II) (Woodgett and Cohen, 1984) with the Myb sequence identified as an in vitro substrate of GSK-3 (Boyle et al., unpublished data) and the c-Jun sequence predicted to be an in vitro substrate of GSK-3. (B) Tryptic digests of HeLa TK- cell c-Jun phosphorylated in vivo (c-Jun) (,',,300 cpm), bp55 c'Jun phosphorylated with GSK-3 in vitro (trpE-c-Jun) (,'.,200 cpm), or an equal mixture of both digests (mix) (,~150 cpm of each digest) were resolved in two dimensions as described in Experimental Procedures and the legend to Figure 1. Purified bp55 c'Jun (",'100 ng) was phosphorylated in vitro in 20 pl of kinase buffer using 1 unit of GSK-3 and 10 p_Ciof [y-32P]ATP(4500 Ci/mmol). The plates were exposed to preflashed film at -70°C for 3 days using an intensifying screen. A tryptic phosphopeptide map of bp16c'Jun(1500 cpm) was carried out in the same manner (right). Purified bp16c'Jun(",,200 ng) was phosphorylated in vitro in 10 ILl of kinase buffer containing 40 I.tM ATP plus 10 p~Ciof [7-32p]ATPwith 1 unit of GSK-3. The plate was exposed to film for 5 hr at room temperature. (C) bp55c'Jun, bp55F243, or bp75v'Junwas phosphorylated in vitro with GSK-3, purified by gel electrophoresis, and subjected to peptide mapping as in (B). Digests of bp55c'Jun(trpE-c-Jun) (~100 cpm), bp55F243 (trpE-c-JunF243) (,~,100cpm), a mixture of bp55c-Gunand bp55F243 (mix) (,~,50 cpm each digest), and bp75v'Jur' (trpE-v-Jun) (325 cpm) were analyzed. All plates were exposed to preflashed film at -70°C for 5 days using an intensifying screen.

region of Myb rich in proline residues resembling its target in glycogen synthase (Boyle et al., unpublished data) (Figure 3A). Using the Myb and glycogen synthase sequences as a guide, we identified a candidate GSK-3 recognition sequence, TPPLSP, in human c-Jun located between amino acids 239 and 244 (Figure 3A). GSK-3 efficiently phosphorylated an N-terminally truncated trpE-c-Jun bacterial protein, bp55 c'Jun, encoding the C-terminal DNA-binding and dimerization domains of c-Jun (residues 194-331) fused to the trpE leader sequence (Angel et al., 1988a). Comparative phosphotryptic peptide mapping of c-Jun phosphorylated in vivo with bp55 c-Gun phosphorylated in vitro revealed that GSK-3 phosphorylated sites corresponding to peptides a, b (bl

and b2), and c (Figure 3B). No other major phosphopeptides were detected in bp55 c'Jun, demonstrating that GSK-3 is specific for authentic sites of phosphorylation. GSK-3 also phosphorylated a c-Jun miniprotein, bp16 c-Gun (bp16 c-Jun contains residues 234-331 of c-Jun spanning a short proline-rich region, the basic region, and leucine zipper), which can be purified to homogeneity in a soluble state. Purified bp16 c'aun was phosphorylated by GSK-3 to a stoichiometry of ,~1.7 mol of phosphate per mol (data not shown). Tryptic digestion of 32p-labeled bp16 c'Jun yielded two major peptides, a and b (Figure 3B), which were found to contain P.Ser (peptide b) and both P.Ser and RThr (peptide a), respectively. Tryptic digestion of both c-Jun and bp55 should release

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Figure 4. Location of GSK-3 Phosphorylation Sites in c-Jun and v-Jun Schematic representation of the c-Jun protein (top) divided into its trans-activation and DNA-binding domains. The DNA-binding domain is a tripartite structure containing a proline-rich region phosphorylatable by GSK-3, the variable basic region, and the heptad leucine repeat sequence (Landschulz et al., 1988). Shown below is a comparison of the human c-Jun tryptic peptide phosphorylated by GSK-3 (amino acids 227-253) with the homologous regions of murine c-Jun (Lamph et al., 1988; Ryder et al., 1988), chicken c-Jun (Nishimura and Vogt, 1988) and v-Jun (Maki et al., 1987), murine JunB (Ryder et al., 1988), and murine JunD (Hirai et al., 1989) proteins. Descending arrowheads mark trypsin cleavage sites. The predicted and deduced phosphoacceptors are boxed, and their residue numbers for human c-Jun are given above the sequences. The amino acid differences between c-Jun and v-Jun, JunD, and JunB are indicated in bold type.

a single peptide located between residues 227 and 252 that contains the predicted GSK-3 recognition sequence, yet 32p-labeled bp55c'Jun yielded three phosphotryptic peptides. This could be explained, however, by the 227252 peptide being phosphorylated at multiple sites. In our two-dimensional system, phosphopeptide isomers run as a series of discrete peptides that lie on a diagonal line with a positive slope (Boyle et al., 1991). Peptides a, b, and c have this characteristic. The presence of both P.Ser and PThr in peptide a (Figure 1B) and the fact that V8 protease digestion of peptides a and b from phosphorylated bp16c'Jun yielded a common RSer-containing peptide are also consistent with the notion that they indeed arise by multiple phosphorylation of a single peptide (data not shown). To confirm that peptides a, b, and c are phosphoisomers arising from residues 227-252, we analyzed bacterially expressed Jun proteins that differ from wild-type c-Jun by a mutation of Ser-243 to Phe (bp55F243). bp55F243 phosphorylated by GSK-3 yielded only two new peptides, b' and c', with electrophoretic mobilities identical to those of b and c, but migrating faster during chromatography, consistent with the substitution of Ser with the more hydrophobic Phe. v-Jun differs from c-Jun in that it contains a deletion in its N-terminus relative to c-Jun, as well as several point mutations throughout its sequence (Maki et al., 1987; Angel et al., 1988a). However, within the region ho-

mologous to peptide 227-252 in human c-Jun it is identical except for a Ser to Phe change at the position equivalent to Ser-243. Phosphorylation of a purified bacterial trpE-v-Jun fusion protein, bp75v'Jun (Angel et al., 1988a), by GSK-3 yielded a peptide map identical to that of bp55F243 (Figure 3C). Digestion of peptide a with V8 protease generated a P.Ser- and PThr-containing peptide from which free phosphate was released during the first cycle of manual Edman degradation (data not shown). There is only one peptide that would be generated by combined tryptic and V8 digestion located within this region of c-Jun that contains both serine and threonine and has a phosphorylatable residue in position 1, namely T_PPSPLIDE. Therefore, this identifies Thr-239 as the site of Thr phosphorylation. Together these results indicate that the sites of GSK-3 phosphorylation in c-Jun are localized to tryptic peptide 227-252, with peptide a being triply phosphorylated, peptide b being doubly phosphorylated, and peptide c being singly phosphorylated. The loss of the triply phosphorylated form in the F243 mutant identifies Ser-243 as a phosphorylation site. Peptide b2, detected in c-Jun from HeLa cells (Figure 1B) and weakly detected in bp55 c'Jun phosphorylated by GSK-3 in vitro (Figure 3B), is therefore likely to be a phosphoisomer in which different phosphorylation sites are occupied in the doubly phosphorylated form of this peptide. Based on their phosphoamino acid compositions, peptide bl could be phosphorylated on two serines (e.g., Ser-243 and -249), and b2 on one serine and one threonine (Thr-239). GSK-3 Phosphorylstion Inhibits c-Jun Binding to the TRE In Vitro The GSK-3 phosphorylation sites are located in a region of c-Jun implicated in binding to the TRE (Figure 4). To test the effect of GSK-3 phosphorylation on c-Jun homodimer binding to the TRE, the activity of bp55 c-Jun purified by TRE affinity chromatography was assayed by gel retardation using a fragment of the collagenase promoter that contains the AP-1 binding site (Angel et al., 1987b) (Figure 5A). Addition of GSK-3 to the reaction did not significantly affect bp55c-Jun homodimer binding, nor the mobility of the DNA-bound complex. However, upon addition of GSK-3 plus ATP, the ability of the homodimer to bind the TRE was reduced by >10-fold. Autophosphorylated GSK-3 alone did not bind to the TRE, indicating that inhibition of bp55 c'Jun DNA binding is not due to a competitive interaction with its substrate, nor did incubation of bp55c°Junwith ATP in the absence of GSK-3 result in the loss of binding activity (data not shown). Because we were concerned that the ,'~37 kd trpE leader sequence in bp55c-Jun might affect its DNA binding, we also tested affinity-purified bp16c'Jun. The addition of ATP alone, over a variety of concentrations ranging from 1 ~M to 1 mM, had no effect on bp16c'Jun DNA binding, but GSK-3 phosphorylation strongly inhibited bp16Jun binding to the TRE site of the collagenase promoter region (Figure 5B). The same result was obtained using an oligonucleotide containing a consensus AP-l-binding site (Figure 5C). The F243 mutant bp16c.Jun was phosphorylated

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Figure5. GSK-3PhosphorylationInhibitsc-Jun Bindingto the TRE The c-Jun bacterialproteinsbp55c'Junand bp16c'Junwere purified to homogeneityby TRE oligonucleotidechromatographyand then used in a gel retardationassayto determinethe effectof GSK-3phosphorylation on DNA binding as described in ExperimentalProcedures. bp55c'Jun and bp16c'Jun were pretreatedat 30°(3 in the absence or presenceof GSK-3or GSK-3and 100 p.M ATP as indicatedat the top of each panel and mixedwith labeledTRE probes,and DNA-protein complexeswere resolvedon nondenaturingacrylamidegels, which weredriedand exposedto film at roomtemperature.In (A) and (B) the probe used was the 173 bp fragment from the 5' region of the collagenasegene, which containsthe AP-1 site, while in (C) the probe usedwas the 22 bp TRE consensusoligonucleotide.The migrationof the boundc-Jun homodimer(Jun), a nonspecificcomplex(NS), and the labeledtarget DNA (P) are marked by arrowheads.

in vitro at two out of three sites, and this was sufficient to result in partial inhibition of DNA binding (data not shown). When affinity-purified bp16c-Jun was phosphorylated to a submolar stoichiometry in vitro using GSK-3 and [7-32p] ATP and passed over a TRE affinity column, and bound protein eluted with 0.4 M KCI, we found that the 32p-labeled bp16c'Jun was present only in the flowthrough fractions, whereas an immunoblot of the same fractions showed that most of the bp16c'Jun in the in vitro kinase reaction was in the fraction eluted at 0.4 M KCI (data not shown). This indicates that the phosphorylated form of c-Jun cannot bind tightly to the TRE and suggests that c-Jun homodimers present in affinity-purified AP-1 are not phosphorylated at the GSK-3 sites. In keeping with this conclusion, peptide maps of in vivo 32p-labeled AP-1 isolated by TRE affinity chromatography from HeLa cells lacked peptides a, b, and c (W. J. Boyle and F. Mercurio, unpublished data).

Serine 243 Is a Site of Negative Regulation by Phosphorylation In Vivo The ability of GSK-3 to inhibit c-Jun DNA binding raised the possibility that c-Jun exists in a latent, phosphorylated form in resting cells, and therefore that expression of a nonphosphorylatable form of c-Jun should increase its trans-activation ability. To test this we analyzed a c-Jun

Ser-243 to Phe mutant (F243), which should resemble a partially dephosphorylated form of c-Jun. Wild-type and mutant c-Jun cDNAs were expressed from the pRSV eukaryotic expression vector (Angel et al., 1988a) in transient transfection assays. Exponentially growing F9 cell and primary rat embryo fibroblast (REF) cultures were transfected with equal amounts of pRSV-c-Jun (WT) or pRSV-cJunF243 (F243) DNA. Immunoprecipitation of mock- or c-Jun-transfected 35S-labeled cells showed that mocktransfected REFs or F9 cells contained little or no endogenous c-Jun protein, whereas in WT- or F243-transfected cultures the predicted 40 kd c-Jun proteins were expressed (Figure 6A). The F243 mutant product was consistently expressed at about 20% the level of its wild-type counterpart, regardless of the cell type used (Figure 6A). Peptide mapping of 32p-labeled WT and F243 c-Jun proteins expressed in transfected REF cultures showed that the WT product was phosphorylated on the major peptides previously identified on human c-Jun in epithelial and fibroblastic cells, including peptides a, b, and c (Figure 6B, left). The F243 protein was phosphorylated on the major sites x and y, but not on peptides a, b, and c (Figure 6B, right). Thus, surprisingly the F243 mutant c-Jun is not phosphorylated at any of the three GSK-3 sites for reasons that are not yet understood. Since the F243 product was not phosphorylated within the DNA-binding domain, we could determine the effect of phosphorylation at these sites on trans-activation efficiency. REF cultures transfected with various amounts of either WT or F243 expression vectors together with the - 7 3 coUagenase-CAT reporter plasmid (Angel et al., 1988a; Chiu et al., 1988) were harvested to measure the level of CAT activity. The F243 c-Jun was consistently a better trans-activator than WT c-Jun when compared in parallel. Whereas 0.5 I~g of the WT expression vector was saturating, trans-activation by the F243 c-Jun increased in a linear fashion with input of vector (Figure 6C). At 1.5-2 p.g of either expression vector, trans-activation by F243 c-Jun was approximately 4-fold higher than by WT c-Jun. Compensating for the underexpression of F243 c-Jun protein relative to WT c-Jun, we estimate that the F243 mutant is at least a 10-fold better activator in vivo. As further evidence that dephosphorylation of c-Jun can activate DNA binding, we have found that AP-1 activity in crude nuclear extracts from serum-starved HeLa cells as measured by gel retardation of TRE-containing oligonucleotides was increased ,~5-fold upon treatment with either calf intestinal alkaline or potato acid phosphatase (data not shown). This effect was abrogated by the phosphatase inhibitor p-nitrophenyl phosphate. In these same cells, we found an ,~,5-fold increase in TRE-binding activity upon treatment with TPA, in agreement with previous estimates of TPA-induced increase in AP-l-binding activity (Angel et al., 1987b; Chiu et al., 1987). These results support the notion that dephosphorylation of c-Jun is a prerequisite to DNA binding, although we cannot be sure that c-Jun is the only target for dephosphorylation in this crude system. Satake et al. (1989) found that acid phosphatase treatment of PEBP1, the presumed mouse AP-1, resulted in loss of binding activity, but this treatment also led to the

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10

pg of expression vector

Figure 6. Phosphorylationand Trans.Activation of Wild-Typeand 1=243 Mutant c-Jun In Vivo (A) Comparisonof c-Jun immunoprecipitatesobtainedfrom REFsand F9 cells transientlytransfectedwith 7 p.gof either RSV-c-Jun(WT) or RSV-c-JunF243 (SF) then metabolicallylabeled with [3SS]methionine/cysteine. Immunoprecipitateswere analyzedby SDS-polyacrylamidegel electrophoresis,fluorographed,and exposedwith an intensifying screen for 7 days at -80°C. the 1=243mutant c-Jun migrates slightly fasterthan the WT coJun.A similardifferencein mobilitywas observedfor F243 bp16c-Juncomparedwith WT bp16¢'Jun. (B)Two-dimensionalphosphotrypticpeptidemappingof c-Junproteins from transientlytransfected REFs metabolicallylabeledwith [32p]orthophosphate.An equivalentnumberof dpm (80-100) of a tryptic digest of each productwas analyzedby two-dimensionalseparationas describedin the legendto Figure2, thenexposedto presensitizedfilm with an intensifyingscreenfor 10 days at -80°C. (C) Trans-activation of WT and F243 c-Jun in REFs. REFswere transiently transfected with the indicated amounts of either RSV-c-Jun (open boxes)or RSV-c-JunF243(hatched boxes), and 2 p,g of the reporter plasmid -73 collagenase-CAT.Resultsfrom three independenttransfectionexperimentswerestandardized(foldtrans-activation) relativeto cells mocktransfectedwith an equivalentamountof pUC18 DNA and reporterplasmid.

Activation of PKC increases the DNA-binding activity of AP-1 and induces the transcription of AP-l-responsive genes even in the absence of de novo protein synthesis (Imbra and Karin, 1987; Angel et al., 1987b, 1988b; Chiu et al., 1987). We have shown here that PKC activation leads to the rapid dephosphorylation of at least one of three phosphorylation sites nested within the c-Jun DNAbinding domain. Phosphorylation of c-Jun protein in vitro by GSK-3 at these three sites severely reduces c-Jun homodimer DNA-binding activity for the TRE. This suggests that in resting cells at least part of c-Jun exists in a fully phosphorylated, latent form incapable of DNA binding. This coupled with the finding that mutation of Ser-243 to a nonphosphorylatable Phe residue inhibits phosphorylation of these sites and activates c-Jun protein in vivo raises the possibility that dephosphorylation of the c-Jun DNA-binding domain is one of the molecular mechanisms underlying PKC-induced activation of AP-1 in vivo. We have shown that Ser-243 and Thr-239 are two of the three sites phosphorylated in the DNA-binding domain of c-Jun (Figure 4). It seems likely that Ser-249 is the third phosphorylation site, although we do not have direct evidence for this. We cannot yet assess the contributions of the individual phosphorylation sites toward the inhibition of c-Jun DNA binding. TPA treatment does not cause a complete dephosphorylation of c-Jun, yet increases AP-1 DNA-binding activity significantly. It is technically difficult to determine which sites are occupied under any given circumstance, but the decrease in the triply phosphorylated form of the 227-252 peptide upon TPA treatment suggests that Thr-239 may be an important site, since peptide a is the major P.Thr-containing form of this peptide. This would also be consistent with our finding that in vitro phosphorylation of bp16c-Gun F243 partially inhibits DNA binding. Ser-243 may also be an important site, but since mutation of Ser-243 causes decreased phosphorylation at all three sites in vivo, we cannot assess its individual contribution. We do not know the overall stoichiometry of phosphorylation of c-Jun at the 227-252 sites in resting cells, but we suspect that it is high, and TPA treatment may lead to an increase in the population of c-Jun lacking phosphate at all three sites. We are now assessing the contributions of each site by site-directed mutagenesis. Comparison of c-Jun in this region with other Jun family members indicates that this sequence is highly conserved. All the Jun proteins contain all three phosphorylation sites, with two exceptions. First, there is a Ser to Asp substitution in the C-terminal serine of this region of mammalian JunB (Ryder et al., 1988) (Figure 4). This Asp might mimic a P.Ser residue, and it would be interesting to determine the effect of mutation of this residue on the DNAbinding properties of JunB. Second, the v-Jun protein of ASV 17 has a Phe in place of the Ser residue found in the chicken c-Jun protein (Maki et al., 1987; Nishimura and Vogt, 1988) at the middle phosphorylation site. Could this

Cell 580

mutation contribute to the oncogenicity of v-Jun? Consistent with this idea, the F243 mutant c-Jun is not phosphorylated at any of the 227-252 sites in REFs, and its trans-activating potential is increased at least 10-fold in a transient assay. However, v-Jun differs from c-Jun in having an N-terminal deletion, as well as three single amino acid changes (Nishimura and Vogt, 1988). Direct analysis to determine which mutations present in v-Jun are required to activate avian c-Jun suggests that deletion of N-terminal sequences is an important determinant in v-Jun-induced transformation of chicken embryo fibroblasts (Bos et al., 1990). The phenotype of the Set to Phe mutation was not tested individually, but the three point mutations together appear to have only a small effect on the focus-forming activity of v-Jun in culture (Bos et al., 1990). However, the effect of these point mutations on tumorigenesis in vivo was not assessed, and v-Jun, like pp60 v-src, may have accumulated a series of independent changes that contribute to the overall oncogenicity of the v-Jun protein. In this regard it should also be noted that the Cys to Ser mutation in v-Jun has now been shown to lie at a site that also negatively regulates c-Jun function in vitro, apparently through oxidation (Abate et al., 1990c). Together these results indicate a potential role for this phosphorylation site mutation in deregulating the function of c-Jun. The conservation of these sites in other Jun family members suggests that these proteins may be regulated by phosphorylation. In this regard, we note that a similar sequence is not present in other structurally related transcriptional regulators, such as C/EBP and CREB (Landschulz et al., 1988; Gonzalez and Montminy, 1989), but all three sites are conserved in the yeast transcriptional regulator GCN4 (Hinnebusch, 1984), which has DNA-binding specificity similar to that of c-Jun. How does phosphorylation of three sites within the c-Jun DNA-binding domain inhibit DNA binding? As mentioned earlier, factors that affect either the stabilities of the c-Jun-c-Jun homodimer or c-Jun-c-Fos heterodimer, or their affinities for DNA, could be involved in regulating AP-1 activity. Two possible models are that phosphorylation interferes with DNA binding without affecting dimerization, or phosphorylation prevents dimerization and acts as an "unzipper" The model proposed for related DNAbinding proteins, including C/EBP, CREB, c-Jun, and c-Fos, that dimerize via a leucine zipper-mediated noncovalent interaction (Landschulz et al., 1988; Vinson et al., 1989) predicts that the variable basic region of each protein within the dimer branches away from the dimerized a-helical coils to form a DNA-binding pocket with its symmetrically opposed arms. Phosphorylation of sites located immediately N-terminal to this region of c-Jun may drastically affect the structural integrity and/or the charge of the DNA binding "pocket" and therefore alter its affinity for the TRE. Since the c-Jun-c-Fos heterodimer is stable in a variety of chaotropic agents, such as high salt and 2.5 M urea, and at elevated temperatures (Smeal et al., 1989), it seems unlikely that its formation would be affected by phosphorylation. However, the c-Jun-c-Jun homodimer is less thermostable (Smeal et al., 1989), and may be sensitive to multiple phosphorylations within the DNA-binding

domain. At present the exact mechanism of inhibition of c-Jun DNA binding by phosphorylation remains to be elucidated. We do not know which protein kinase phosphorylates c-Jun at the 227-252 sites in vivo. GSK-3 was originally described as a soluble cytoplasmic enzyme involved in the regulation of glycogen metabolism in skeletal muscle (Hemmings et al., 1982), perhaps arguing that GSK-3 is not a c-Jun protein kinase. In addition, GSKo3 has been localized predominantly to the cytoplasm in fibroblasts (J. R. W., unpublished data), which would preclude its phosphorylation of c-Jun in the nucleus. However, the primary structure of two GSK-3 isozymes has recently been determined by molecular cloning of two highly related genes, ~ and 13(Woodgett, 1990), and there is a 75%-80% identity between GSK-31~ and the newly reported sequence of the zeste-white3 (zw3) gene of Drosophila melanogaster (Siegfried et al., 1990), which is a segment-polarity gene with pleiotropic effects in both the embryo and adult that is responsible for epidermal cell fate determination. zw3 thus appears to be the Drosophila homolog of mammalian GSK-31~. By analogy with the role of zw3 in segment organization and cell fate determination, both of which are likely to involve transcriptional regulation of downstream genes, a role for GSK-3 in the negative regulation of c-Jun is plausible. However, even without being able to prove that GSK-3 acts in vivo as a c-Jun protein kinase, GSK-3 has been an important experimental tool that has allowed us to manipulate in vitro the phosphorylation state of physiologically relevant sites on c-Jun. There are other aspects to the regulation of AP-1 activity by phosphorylation. For instance, we do not yet know the consequences of the GSK-3 phosphorylations on the ability of the c-Fos-c-Jun heterodimer to bind the TRE. Moreover, c-Fos itself is phosphorylated at multiple sites (Barber and Verma, 1987), and these phosphorylations may be important in regulating DNA binding (ML~lleret al., 1987). Finally, in addition to the 227-252 phosphorylation sites in c-Jun, there are at least two other sites of phosphorylation in c-Jun (peptides x and y), which lie outside the minimal DNA-binding domain but have not yet been mapped precisely. The function of these sites is not known, but it is possible that these may regulate the function of c-Jun in a positive fashion. For instance, their phosphorylation could increase the trans-activating ability of c-Jun in a manner analogous to the activation of CREB by PKA, and be a target for mitogen-activated protein kiRases. Phosphorylation of c-Jun is clearly not the only means of regulating AP-1 activity. Another pathway leading to increased AP-1 activity in response to PKC activation occurs through c-fos induction (Greenberg and Ziff, 1984). Activation of PKC leads to the rapid and transient expression of c-Fos, which forms heterodimers with c-Jun or Jun-related proteins, such as JunB and JunD, that are capable of binding the TRE (Nakabeppu et al., 1988). However, several lines of evidence indicate that this mechanism cannot fully account for the rapid induction of AP-1 activity in vivo as measured by autoinduction of c-jun expression through its TRE (Angel et al., 1988b). First, c-fos can be induced

PKC ActivationDephosphorylatesc-Jun 581

by elevating cAMP levels, but we found that this treatment does not induce c-jun or AP-1 activity (Chiu et al., 1989) nor does it modulate c-Jun phosphorylation. Second, in undifferentiated F9 teratocarcinoma cells TPA induces c-los expression, whereas c-jun expression is refractory to this treatment (Yang-Yen et al., 1990). Conversely, when the same cells are differentiated by retinoic acid, c-jun expression is induced upon TPA treatment in the absence of c-fos expression (Yang-Yen et al., 1990). Third, treatment of HeLa cells with a low dose of staurosporine inhibits the induction of c-los by TPA without exerting any effect on the induction of c-jun (Y. Devari and M. Karin, unpublished data). Thus, the autoinduction of c-jun is responsive to PKC activation without the need for either preexisting or concomitant expression of c-los. Furthermore, both quiescent HeLa cells and human fibroblasts express very low levels of junB and junD mRNAs prior to or after 60 min of TPA treatment (Chiu et al., 1989) and we do not detect c-Fos or Fos family members associated with c-Jun in uninduced HeLa cells (J. Meek, Y. Devari, and M. Karin, unpublished data). The rapid induction of AP-1 activity in the cells we examined is therefore unlikely to occur by modification of preexisting JunB or JunD, or Jun-Fos heterodimers, suggesting that the main target for the PKC pathway is c-Jun. Nevertheless, the extent to which posttranslational events are involved in the early induction of AP-1 activity will undoubtedly vary from cell to cell, and depend on the basal level of c-Jun in the cell in question. In addition, at longer times after TPA stimulation, newly synthesized c-Fos and c-Jun will become major contributors to the increase in AP-1 activity. Although it has been proposed that regulation of gene expression involves changes in protein phosphorylation, there have been few reports that phosphorylation of transcription factors modulates their activity directly. Phosphorylation of the yeast transcriptional activator ADR1 by PKA inactivates its function (Cherry et al., 1989), without affecting DNA binding. Like c-Jun, the DNA-binding activity of c-Myb for a synthetic consensus binding site is inhibited upon phosphorylation by casein kinase II in vitro (LLischer et al., 1990), but there is no direct evidence that this affects c-Myb activity in vivo. There are also examples where phosphorylation stimulates transcription factor activity. The DNA-binding activity of the serum response factor requires phosphorylation by casein kinase II (Manak et al., 1990). The DNA-binding activities of the E2F and E4F transcription factors induced by adenovirus infection are reportedly increased by phosphorylation (Bagchi et al., 1989; Raychaudhuri et al., 1989). In the case of CREB, phosphorylation does not affect DNA binding, but apparently affects the ability of CREB to interact with other elements of the transcription apparatus (Gonzalez and Montminy, 1989). The same is true for heat shock factor (Sorger and Pelham, 1988). Phosphorylation of two N-terminal serine residues by PKA or PKC is required for v-ErbA to suppress transcription of erythroid-specific genes (Glineur et al., 1990). Phosphorylation of GAL4 is correlated with its ability to activate transcription (Mylin et al., 1990), and the phosphorylation of

Spl by the double-stranded DNA-dependent protein kinase is increased when Spl binds to its target DNA (Jackson et al., 1990). Finally, IKB, an inhibitory protein that binds to and sequesters the NF-KB transcription factor in the cytoplasm, is inactivated by phosphorylation (Shirekawa and Mizel, 1989; Ghosh and Baltimore, 1990). Thus, protein phosphorylation can exert either positive or negative effects on transcription factors. Conclusions

We propose that at least part of the c-Jun protein is present in resting cells in a latently active phosphorylated state that is recruited for DNA binding upon dephosphorylation. The observed site-specific dephosphorylation of c.Jun in response to the activation of PKC suggests that c-Jun is regulated like glycogen synthase. Thus c-Jun would be phosphorylated at the 227-252 sites by a constitutively active protein kinase. Upon activation of PKC, a protein phosphatase activity would be elevated, leading to net dephosphorylation of the 227-252 sites, which in turn would result in c-Jun activation. Alternatively, PKC activation may cause inhibition of the c-Jun protein kinase, resulting in a net dephosphorylation brought about by a constitutively active phosphatase. The regulation of c-Jun phosphorylation may be the final step in the pathway initiated by activation of PKC, which leads to increased AP-1 activity. Identification of both the protein kinases and phosphatases that act antagonistically to modulate c-Jun function is of obvious importance, as is an understanding of their regulation in vivo. Experimental Procedures

Materials The followingmaterialswere used in this study: TPCK-trypsin(WorthingtonBiochemicalCorp.)(storedas a I mg/rnlsolutionin 0.1 mM HCI at -196°C); S. aureus V8 protease (ICN Immunochemicals);TPA (SigmaChemicalCo.)(storedas a 5 mg/ml stock solutionin absolute ethanolat -20°C priorto dilution);potatoacid and calf intestinalalkaline phosphatases(BoehringerMannheimBiochemicals).GSK-3was purified from rabbitskeletalmuscle as described(Woodgettand Cohen, 1984). Cell Culture, Cell Labeling, and Immunopreclpltatlon

The HeLa thymidinekinase negative(TK-) cell line (humancervical epidermal carcinoma), MG63 cell line (human osteosarcoma),and Flow 2000 cells (normal embryonic human lung diploid fibroblasts) were grown in Dulbecco'smodifiedEagle'smedium(DMEM)containing 10% fetal bovine serum (FBS) (Hyclone Laboratories)and lx nonessentialamino acid mixture (Difco Laboratories).Adherentcell cultures were labeledwith either 0.5 mCi/ml 35S-labeledaminoacids (Tran35S-label,ICN Radiochemicals),or 3.0 mCi/ml [32p]orthophosphate(ICN Radiochemicals)in medialackingeitherL-mathionineand L-cysteine or lacking sodium phosphate. Prior to adding label, exponentiallygrowingconfluentcultures were serum starvedfor 24-48 hr in 0,5% FBS and then washedtwice with Tris-buffaradsaline.Cultures were incubatedfor 30 rain in medialackingeither L-methionina and L-cysteineor sodiumphosphate,labelswereadded,and thenthe cultures were incubatedfor another3 hr. To some cultures, TPA was addedto a final concentrationof 100 ng/ml for the final 15-60 rain of cell labeling. Labelingmediawere removedand the cultureswerewashedtwice with ice-coldTris-buffaredsaline,then very brieflywith ice-coldsterile water.Nucleiwereisolatedfromcells afterlysis in hypotonicbuffer(20 mM HEPES-NaOH[pH 7.4],5 mM KCI,5 mM MgCI2,5 mM dithiothreitol, 0.1 mM EDTA,50 mM NaF,0.1% aprotinin,0.1% NP-40)as previ-

Cell 582

ously described (Chiu et al., 1988). Crude nuclei were collected after centrifugation at 1000 x g for 10 rain then washed once in nuclear isolation buffer. To solubilize c-Jun protein completely, the washed nuclei were pelleted and then lysed in 100 ml of boiling-hot SDS-lysis buffer (Chiu et al., 1988). The lysatas were diluted to 1.0 ml with ice-cold RIPA buffer (Hunter and Sefton, 1980) and DNA was sheared using a 20gauge needle to reduce viscosity. The lysates were then clarified by centrifugation at 10,000 x g for 30 min at 4°C. Total c-Jun protein was immunoprecipitatad from cell lysatas using polyclonal anti-c-Jun antiserum as described (Chiu et al., 1988). Washed immunoprecipitatas were solubilized in Laemmli sample buffer and then resolved on 12.5% SDS-polyacrylamide gels. Labeled proteins were visualized by direct autoradiography of untreated dried gels at -70°C using presensitized film and an intensifying screen. Mlcroanalysls of c-Jun Phosphoprotein c-Jun protein phosphorylated in vivo or in vitro was fractionated on SDS-polyacrylamide gels and detected by autoradiography. Phosphorylatad proteins were elutad from excised gel bands as described (Hunter and Sefton, 1980). Protein carrier (20 p.g of RNAase A) was added to the eluate, and proteins were precipitated with 20% TCA for' 1 hr at 0°C. TCA precipitates were washed in ethanol and then oxidized in performic acid at 0°C. Samples were then digested to completion with TPCK-treated trypsin. Salt was removed from the diQests by repeated lyophilization, and the digests were applied to thin-layer cellulose plates for two-dimensional peptide mapping (Hunter and Sefton, 1980) by electrophoresis at pH 1.9 for 25 rain at 1.0 kV in the first dimension, and then by chromatography in phosphochromo buffer (Hunter and Sefton, 1980) in the second dimension as described elsewhere (Boyle et al., 1991). The phosphoamino acid composition of c-Jun protein and individual tryptic peptides was determined by twodimensional electrophoresis of partial acid hydrolysates as described (Hunter and Sefton, 1980). The strategy to identify the position of phosphoamino acid residues in V8 subfragments of tryptic phosphopeptide a using manual Edman degradation has been previously described (Hunter et al., 1984).

Purification of Jun Proteins Expressed In E. coil The trpE-c-Jun bacterial protein bp55 c-Junwas expressed in E. coil as described (Angel et al., 1988a; Chiu at al., 1988). Purification, renaturation, and phosphorylation of trpE fusion proteins will be described in detail elsewhere (Boyle et al., unpublished data). Renatured bp55 c-Ju" was affinity purified by TRE oligonucleotide chromatography as described (Angel st al., 1987b), and then dialyzed in 20 mM HEPESNaOH (pH 7.4), 60 mM KCI, 5 mM dithiothreitol, 0.1 mM EDTA, and 5% glycerol. To express a soluble form of the c-Jun DNA-binding domain, the Aval to BamHI fragment of the human c-jun cDNA clone encoding residues 234-331 was ligated in frame with the start codon of the T7 expression vector pET-3 (Studier et al., 1990). The resulting plasmid, pT7cjunAva, was transfected into BL21(DES)IpLysS, and its expression product, bp16c-Jun, was induced at ODsoo ~0.6 in the presence of 0.4 mM IPTG at 28°C for 2 hr. bp16c-Jun was partially purified from soluble extracts by precipitation with 40% saturated ammonium sulfate at 0°C for 1 hr to remove an endogenous nuclease, then further purified to homogeneity by TRE oligonucleotide chromatography (Angel et al., 1987b). Pn~teln Klnaae Assays Purified c-Jun (100-200 ng) was diluted in 20 I~1of ice-cold protein kinasa buffer containing 20 mM HEPES-NaOH (pH 7.4), 10 mM MgCI2, 1.0 mM dithiothreitol, and 0.1 mM EDTA including 40 p.M ATP (10 p.Ci [7-32p]ATR 4500 Ci/mmol, ICN Radiochemicals). Protein kinase reactions were initiated by the addition of approximately 1 unit of the relevant protein kinase and incubated at 30°C for 10-15 min. For reactions using PKC, Ca 2+ and emulsified lipid were added as previously described (Woodgett and Hunter, 1986). Site-Directed Mutageneals of c-Jun The c-Jun F243 mutant was created by substituting the codon for Ser243 with a codon for Phe by site-directed mutagenesis (Zoller and Smith, 1988). Briefly, a fragment of the human c-jun cDNA extending from the internal Pstl to BamHI restriction sites (Angel et al., 1988a) was subcloned into pBluescript to generate single-stranded template

DNA. Ser-243 was mutated to Phe using the 29 bp oligonucleotide shown below: Phe 5'-CCG CCC CTG TTT CCC Arc GAT ATG TC-3'. Recombinant mutant clones were identified by colony hybridization and then sequenced using the dideoxy nucleotide chain termination method to confirm the presence of the mutation. The Aval to BamHI fragment was subcloned into the pTrpE.cJ (Angel et al., 1988a) or pET-3 (Studier et al., 1990) bacterial expression vectors and resequenced to confirm the presence of the Ser-243 to Phe mutation.

DNA-Blndlng Assays Recombinant c-Jun DNA-binding activity was measured by gel retardation as described ($meal et al., 1989). Briefly, protein DNA complexes were formed at either 4°C or 23°C for 30 min in 10-20 p.I of 12 mM HEPES-NaOH (pH 7.9), 4 mM Tris-HCI (pH 7.9), 60 mM KCI, 30 mM NaCI, 5 mM MgCI2, 5 mM dithiothreitol, 0.1 mM EDTA, and 12.5% glycerol containing 100 mg/ml bovine serum albumin, 100 p.g/ml poly(dl-dC), and 0.1 ng of 32p end-labeled TRE-containing DNA (,~,1.0 x 109 cpm/l~g). The target DNA was either the 173 bp Pvul to EcoRI fragment of pTRE-TK-CAT containing the TRE of the collagenass gene (Angel et al., 1987b; Smeal st al., 1989), or the following doublestranded TRE-containing oligonucleotide: 5~AGCTTGGTGACTCATCCG-3 3~ACCACTGAGTAGGCCTAG-5

' '.

Specificity of binding was determined by substrate binding competition using both unlabeled wild-type and mutant (D-72 TRE) collagenase TRE sequences and by preincubation with anti-c-Jun antibodies (Chiu et al., 1988). Protein-DNA complexes were resolved on 5% nondenaturing polyacrylamide gels (acrylamide:bisacrylamide equal to 80:1) containing 0.25x TBE buffer (pH 7.9) at 200-250 V for 2.5-3 hr at 4°C. Prior to the DNA-binding assay, proteins (100-200 ng) were modified by phosphorylation in 5 pJ kinase reactions, as described above, at 30°C for 10 min, then chilled to 4°C.

Transient Expression of Wild-lype and F243 Mutant c-Jun The Pstl to Notl fragment of the mutated c-Jun pBluescript plasmid was subcloned into the pRSV-c-Jun expression vector (Angel et al., 1988b) and used together with the parent pRSV-c-Jun for transient expression of mutant and wild-type proteins (Angel st al., 1989; Smeal et al., 1989). Primary REFs were prepared from day 14 Fisher rat embryos and grown in DMEM supplemented with 10% FBS. F9TK- cells were grown in F12-DMEM (1:1) supplemented with 10% FBS and 0.1 mM {~-msrcaptoethanol. Two to four hours prior to transfection, fresh medium was added to exponentially growing cell cultures plated on 10 cm plates. The cells were incubated with 0.5-2 pg of c-Jun expression vector and 2 p~gof reporter plasmid coprecipitatad with calcium phosphate for 8-10 hr, then rinsed with fresh medium. Cells were harvested 16 hr later, and lysates were analyzed for induced CAT activity. Expression and phosphorylation of exogenous c-Jun proteins were determined by labeling cell cultures in 10 cm dishes transfected with 7 p.g of c-Jun expression vectors in 2 ml of labeling medium for 3-6 hr prior to harvest with either [32p]orthophosphate (5 mCi/ml; 6 hr labeling) or Tran35S-label (150 pCi/ml; 3 hr labeling) in DMEM lacking either sodium phosphate or L-methionine, respectively, c-Jun proteins were immunoprecipitated from cells lysed in RIPA buffer using a polyclonal anti-c-Jun antibody directed against a C-terminal peptide of human c-Jun, residues 316-331 (VNSGCQLMLTQQLQTF) (Angel et al., 1988a). Acknowledgments W. J. Boyle and T. Smeal made equal contributions to this work. We thank Frank Merourio, Elizabeth Allegretto, and Jennifer Meek for kindly providing purified HeLa cell AP-1 and the TRE oligonuctsotide affinity column; Bernard Binetruy for day 14 REF cultures; Ellen Freed and Peter van der Gear for the MG63 and Flow 2000 cell cultures; and both Jill Meisenhelder and Chris Cartwright for 32p-labeled cell lysates. We also thank Gary Glenn and Walter Eokhart for communicat-

PKC Activation Dephosphorylates o.Jun 583

ing their unpublished data on murine c-Jun phosphorylation and Yoram Devari for his unpublished data on the effects of stau rosporine on c-jun induction. L. H. K. D. was supported by a fellowship from the Dutch Queen Wilhemina Fund, R A. was supported by a postdoctoral fellowship from the Deutscher Akademischer Austauschdienst, and W. J. B. was a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation. This work was supported in part by US Public Health Service grants CA14195, CA39780 (to T. H), CA50528, and ES04151 (to M. K.), a Council for Tobacco Research grant and Department of Energy grant FG03-86ER60429 (to M. K.), and an American Business Foundation Research Award (to T. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC Section 1734 solely to indicate this fact. Received June 20, 1990; revised November 9, 1990.

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By phosphopeptide mapping we have found that c-Jun transiently expressed in F9 cells is underphosphorylated at the 227-252 sites compared to c-Jun expressed in more differentiated cells, which is consistent with its greater trans-activating activity in F9 cells. In addition, v-Jun expressed in F9 cells, like F243 c-Jun, is not detectably phosphorylated at the 227-252 sites.