Affinity-purified c-Jun amino-terminal protein kinase requires serine/threonine phosphorylation for activity.

The addition of phorbol esters to U937 leukemic cells stimulates the phosphorylation of c-Jun on serines 63 and 73. To isolate the protein kinase which stimulates this phosphorylation, we have used heparin-Sepharose chromatography followed by affinity chromatography over glutathione-Sepharose beads bound with a fusion protein of glutathione S-transferase and amino acids 5-89 of c-Jun (GST-c-Jun). Using this procedure we purify a 67-kDa protein which is capable of phosphorylating GST-c-Jun as well as the complete c-Jun protein. By making mutations in serines 63 and 73 and then creating a fusion protein with GST (GST-c-Jun mut), we demonstrate that this protein kinase specifically phosphorylates these sites in the c-Jun amino terminus. Treatment of purified c-Jun amino-terminal protein kinase (cJAT-PK) with phosphatase 2A inhibits its ability to phosphorylate GST-c-Jun. This inactivated enzyme can be reactivated by phosphorylation with protein kinase C (PKC), although PKC is not capable of phosphorylating the GST-c-Jun substrate. Because v-Jun cannot be phosphorylated in vivo, we compared the ability of cJAT-PK to bind to GST-v-Jun or GST-c-Jun mut. The cJAT-PK bound 50-fold better to GST-c-Jun mut than GST-v-Jun suggesting that the delta domain which is missing in v-Jun plays a role in binding the cJAT-PK. These results suggest that there is a protein kinase cascade mediated by protein phosphatases and PKC which regulates c-Jun phosphorylation.

I To whom all correspondence should be addressed. Tel.: 205-934-codon at amino acid 234 and, within this plasmid, mutating serines 63 and 73 to leucines, we have demonstrated that these 2 serines are the major sites of c-Jun phosphorylation in vivo (8). We further found that adding other protein kinase C (PKC)' activators (diacylglycerol and bryostatin), okadaic acid, a phosphatase inhibitor, but not activators of cyclic AMP-dependent protein kinase stimulates the amino-terminal phosphorylation of c-Jun in these cells (8). To examine the role of this phosphorylation in regulating c-Jun-mediated transcriptional activation, we constructed a fusion protein containing the 84 NH2-terminal amino acids of c-Jun and fused it to the DNA-binding domain of the yeast GAL4 protein. Transfection of this plasmid along with a recorder gene demonstrates that this short segment of c J u n protein is sufficient to mediate transcriptional activation by phorbol esters (8). However, mutation of serines 63 and 73 to leucines blocks this activation, suggesting that amino-terminal phosphorylation is necessary if transcriptional activation in U937 cells is to occur. Similarly, these sites in c-Jun have been shown to be phosphorylated by H-ras transfection of F9 cells and to mediate c-Jun transcriptional activity in these cells (9,10). These data suggest that the protein kinases (PK(s)) which mediate the amino-terminal phosphorylation of c-Jun play a critical role in regulating transcription.
Addingphorbol esters to U937 cells also induces an increase in JunB protein. While this member of the Jun family forms a heterodimer with c-Fos and binds to AP-1 sites, it blocks transcriptional activation mediated by c-Jun (11-13). In contrast, adding phorbol esters to U937 cells do not activate phosphorylation of JunB (8). Also, the 89 NHp-terminal amino acids of JunB do not activate the transcriptional activity of the Gal4 DNA-binding domain in U937 cells (8). Although the amino terminus of c-Jun and JunB are highly similar (50%), serines 63 and 73 of c-Jun are followed by prolines; whereas, in JunB they are followed by a serine and aspartic acid residue, respectively. These data suggest that differential phosphorylation of Jun family members may determine their ability to function as either transcriptional activators or inhibitors.
In v-jun, the transforming gene isolated from avian sarcoma virus 17 (14), when compared to c-Jun the sequence surrounding serines 63 and 73 is identical. In comparison to c-Jun, however, v-Jun is deleted of 27 amino acids from position 34 to 60. This region of the c-Jun protein has been shown to bind a potential inhibitor of transcriptional activation (15,16). It is clear that these 27 amino acids are important in the regulation of transformation, since removal of specific amino acids within this region increases the transforming activity of c-Jun (17). Unlike c-Jun, v-Jun is not phosphorylated when u937 cells are treated with phorbol esters, suggesting that the amino-terminal PK might need to bind to the deleted region not found in v-Jun before it can phosphorylate serines 63 and TO better understand how the c-Jun amino-terminal kinase functions, we have isolated this kinase by using heparin-Sepharose chromatography followed by affinity chromatography using the 84 NHz-terminal amino acids of c-Jun linked t o glutathione S-transferase. The purified PK has a molecular mass of 67 kDa. It will phosphorylate the amino terminus of c-Jun but not a JunB or an amino-terminal fragment of Jun with serines 63 and 73 mutated to leucines. It is active in the presence of magnesium and manganese. It is not recognized by antipeptide antibodies to pp42j44 (ERK 1 and 2). Dephosphorylation of this purified enzyme with phosphatase 2A blocks its ability to phosphorylate c-Jun, suggesting that it is activated by serinejthreonine phosphorylation. The ability of this enzyme to phosphorylate c-Jun can be reactivated in uitro by phosphorylation with PKC. This c-Jun phosphorylating P K binds with a higher affinity to c-Jun than v-Jun, suggesting that the 27-amino acid deletion in the amino terminus plays a critical role in the enzyme-binding site.

EXPERIMENTAL PROCEDURES
Isolation of the c-Jun Amino-terminal PK (cJAT-PK)-PMAtreated U937 cells (approximately 10') were washed twice with phosphate-buffered saline, and the cell pellet was resuspended in an equal volume of lysis buffer A (20 mM Hepes, pH 7.5, 1 mM EGTA, 2 mM MgCl,, 2 mM MnC12, 1 mM dithiothreitol, 0.5% Nonidet P-40,0.5 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, 0.6 M NaC1). U937 cells were incubated on ice for 1 h and centrifuged at 1000 X g for 15 min at 4 "C. Glycerol was added to the supernatant to a concentration of 50%. An equal volume of lysis buffer was added to the supernatant to bring the final concentration of NaCl to 0.3 M. This extract was passed over a 1-ml glutathione S-transferase/glutathione-Sepharose column (Pharmacia LKB Biotechnology Inc.). The column was washed with 3 ml of buffer A containing 0.3 M NaCl. The flowthrough and wash were combined and loaded onto a heparin-Sepharose column (Pierce Chemical Co.). The column was washed with buffer A containing 0.3 M NaC1, and the PK activity was eluted with a 20-ml linear gradient of KC1 (0. 3-1.5 M) in buffer A. The flow rate was approximately 0.25 ml/min, and 0.5-ml fractions were collected. Fractions with the highest amino-terminal c-Jun kinase activity were combined and dialyzed for 2 h against buffer A containing 0.3 M KCl. This material was loaded onto a 1-ml Jun (amino acids 5-89)/ glutathione S-transferase/glutathione-Sepharose (see below for details) column and washed with 3 ml of buffer A containing 0.6 M KC1. The proteins were eluted with 3 ml of buffer A containing 80% ethylene glycol. Eluted proteins were dialyzed against buffer A for 2 h and concentrated with 50% polyethylene glycol. Glycerol was added until the concentration reached 50%, and it was stored at -20 "C.
Assay of Amino-terminal c-Jun Kinase Actiuity-Preparation of c-Jun:glutathione S-transferase fusion protein bound to glutathione-Sepharose beads to create a substrate for the cJAT-PK polymerase chain reaction was used to clone amino acids 5-89 of c-Jun, JunB, and c-Jun with serines 63 and 73 mutated to leucines (c-Jun mut) into the BamHI/EcoRI sites of the pGEX-2T vector (22) (Pharmacia LKB Biotechnology Inc.). This vector expresses glutathione S-transferase and contains a thrombin cleavage site between this protein and the fused amino terminus of the Jun fragments. Escherichia coli cells transfected with either the pGEX-Jun fusion or pGEX-2T were stimulated with 0.4 mM isopropyl-1-thio-@-D-galactopryanoside for 4 h. The bacterial pellets were lysed in PBST buffer (150 mM NaC1, 16 mM sodium phosphate, pH 7.5, 1% Triton X-100, 2 mM EDTA, 0.1% IdMeOH. 0.2 mM phenylmethylsulfonyl fluoride, and 5 mM benzamidine), sonicated three times for 30 s on ice, and centrifuged at 10,000 X g for 10 min at 4 "C. Then the supernatant was incubated with an equal volume of 50% (v/v) solution of glutathione-Sepharose beads (Sigma) for 1 h at 4 "C. The beads were washed twice with PBST buffer containing 1.5 M KC1 and twice with PBST without salt. A slurry suspension of beads (50% v/v) in glycerol was stored a t -20 "C.
Measurement of PK Activity-cJAT-PK activity was measured in a 20-p1 reaction containing 0.2 pg of c-Jun/glutathione S-transferasel glutathione-Sepharose beads, 5 p1 of protein extract, and 10 p1 of kinase buffer, 20 mM Hepes, pH 7.6,l mM EGTA, 1 mM dithiothreitol, 2 mM MgC12, 2 mM MnCl,, 5 mM NaF, 1 mM Na vanadate, and 50 cpm/fmol [-Y-~'P]ATP. The reaction was run at 30 "C, and terminated by the addition of 0.5 ml of PBST. The beads were pelleted and washed with cleavage buffer, 20 mM Tris-HC1, pH 7.6, 150 mM NaC1, 2.5 mM CaC12, 0.1% PMeOH. The beads were then treated with thrombin (3.5 ng) and incubated at room temperature for 1 h. Thrombin cleavage was terminated by adding sample buffer, the beads were pelleted, and the supernatant was loaded onto a 15% SDS-polyacrylamide gel. After electrophoresis, the gel was dried and exposed to film overnight.
Reconstitution of c-Jun PK Activity from SDS Gels-The cJAT-PK samples were mixed with equal volumes of SDS sample buffer (2% sodium dodecyl sulfate, 63 mM Tris, pH 6.8, 5 mM EDTA, 10% glycerol, and 5% mercaptoethanol). Samples were electrophoresed on a 8% SDS gel. After electrophoresis the gel was washed twice for 15 min with 50 mM Tris, pH 7.5, 4 M urea, 20 mM EDTA, and 10 mM PMeOH. and twice with 20 mM Hepes, pH 7.6, 0.5 mM EGTA, and 15% glycerol. Slices of the gel were then rotated overnight in the latter buffer. One-tenth volume of the extract was used to assay cJAT-PK activity.
Treatment of c-Jun with Phosphatase 2A and Reconstitution with PKC-Prior to beginning the experiment the cJAT-PK was dialyzed into 20 mM Hepes, pH 7.5, 0.5 mM EGTA, 1 mM dithiothreitol. The cJAT-PK was incubated for 60 min at 37 "C with 20 ng ofphosphatase 2A (a gift of Dr. Marc Mumby, University of Texas Southwestern Medical Center, Dallas, TX). The reaction was terminated by adding NaF to a final concentration of 5 mM. PKC, partially purified from rat brain, was a mixture of cy and isoforms (a gift of Dr. B. Bishop, Schering-Plough Cow. Bloomfield, NJ). After phosphatase treatment 180 ng of PKC was added with and without calcium and phospholipids, as described previously (19). Aliquots of this reaction mixture were then assayed for cJAT-PK activity. The PKC inhibitor containing amino acids 19-36, identical to the pseudosubstrate domain, was purchased from Gibco/Bethesda Research Laboratories.

RESULTS AND DISCUSSION
Because c-Jun is phosphorylated both on amino-and carboxyl-terminal serines (7,8,20), it was necessary to develop an assay which would recognize only the amino-terminal PK.
To accomplish this, 84 amino acids (5-89) which include serines 63 and 73 in the X and Y peptides (7,8) (22), were fused to glutathione S-transferase (GST) which contained a thrombin cleavage site at its carboxyl terminus. The fusion protein was expressed in E. coli and the bacteria lysate was mixed with glutathione-Sepharose beads. To assay PK activity, extracts or column eluates were incubated with the fusion protein beads, [y3*P]ATP, and Mg2' at 30 "C for 10 min. The beads were pelleted, washed, and cleaved with thrombin. The supernatant was then run on a 15% SDS gel to identify the phosphorylated Jun peptide. In partially purified or fully purified extracts from cells no phosphorylation of the GST protein was seen (data not shown).
To isolate the c-Jun amino-terminal PK, U937 cells were first treated with PMA (0.15 UM) for 30 min to activate the PK in vivo and lysed (see "Experimental Procedures") (22). This high salt extract of the cells was passed over a GST/ glutathione-Sepharose column, and the flow-through bound to a heparin-Sepharose column. The active enzyme was eluted with a linear KC1 gradient and the fractions with the highest activity were combined, dialyzed, and loaded onto a substrate affinity column containing the amino-terminal c-Jun GST fusion protein bound to glutathione-Sepharose beads (Fig. lA,  lane 1 ). The column was washed extensively, and the PK was eluted in the same buffer containing 80% ethylene glycol (Fig.  L4, lune 2 ) . The enzyme was 5000-fold purified with a yield of 8-15 ~g / 2 X lo8 cells. Silver staining of an SDS gel containing an aliquot of the final column eluate demonstrates a major band at 67 kDa. When increased protein is loaded  1. A, purification of cJAT-PK. Purification of cJAT-PK was carried out as described under "Experimental Procedures." Aliquots from each stage of purification were run on a 10% SDS gel which was silver stained. Lane I contains the proteins bound to the GSTJun affinity column. Lane 2 contains the concentrated ethylene glycoleluted cJAT-PK. B, reconstitution of cJAT-PK. The ethylene glycol extract from GST:c-Jun beads was concentrated and run on an SDS gel which was sliced. Reconstitution of cJAT-PK activity was carried out as described under "Experimental Procedures" and aliquots of the reconstituted material were assayed for cJAT-PK activity. C, the effect of increasing enzyme concentration on phosphorylation of the GSTc-Jun fusion beads. Increasing amounts of enzyme were added to 20 pg of GST:c-Jun beads in a 10-min PK assay. The reaction was terminated and run on an SDS gel. The autoradiogram of this gel is shown in the insert, and the counts/min in the c-Jun amino terminus are plotted. D, time course of cJAT-PK phosphorylation of GSTc-Jun fusion protein. 2 units of enzyme were added to GSTcJun beads for varying periods of time in a cJAT-PK assay as described under "Experimental Procedures." At each time point the reaction was stopped with 1 ml of PBST. The inset is the autoradiogram of c-Jun amino-terminal peptide. The SDS gel was cut and counted. minor bands are seen between 46 and 69 kDa. When U937 cells which are not treated with PMA are used for purification no cJAT-PK activity was evident.
To identify whether the 67-kDa protein was the c-Jun PK, the PK activity was reconstituted from a SDS gel. After electrophoresis, the gel was washed two times with a urea containing buffer. Slices of the gel were then extracted overnight with a buffer containing 0.5 mM EGTA, and each extract was assayed for c-Jun PK activity. The only PK activity evident was in lane 5 ( Fig. 1B) which correlates to a position at approximately the 69-kDa marker protein. This experiment demonstrates that the purified cJAT-PK is the major band seen on silver stain of the affinity column eluate.
Using purified c-JAT PK, the time and concentration dependence of phosphorylation was examined. Half-maximal phosphorylation of the GST-c-Jun fusion protein at 30 "C was achieved in 8 min (Fig. 1D) and the rate of incorporation appeared linear over 10 min using 2 units (1 unit of cJAT kinase equals 660 cpm/l0 min) of enzyme. Using a 10-min period of incubation, the phosphorylation of the GST-c-Jun fusion protein was linear to 2 units of enzyme (Fig. IC). This amino-terminal c-dun kinase can be activated either in the presence of magnesium or manganese with maximal stimulation occurring at 2 and 1 mM, respectively. Because it is possible that the Jun peptide fused to GST is abnormally folded and becomes a substrate for this PK, we examined whether this PK can phosphorylate the entire c-Jun protein, using bacterially expressed c-Jun . After expression of this c-Jun protein in bacteria, it was solubilized in urea (7), followed by the removal of the urea by dialysis. This bacterially expressed c-Jun protein is phosphorylated by cJAT-PK. (Fig.   2B). To determine whether this cJAT-PK phosphorylates identical serines (63 and 73) to those modified in uiuo, we constructed a GSTamino-terminal Jun fusion protein in which serines 63 and 73 were mutated to leucines (22). If serines 63 and 73 were the only sites of phosphorylation, then this mutant should not be phosphorylated by the cJAT-PK. Because we have shown that addition of phorbol esters to U937 cells does not lead to an increase in JunB phosphorylation, we constructed a GST fusion protein encoding the 89 NH2-terminal amino acids of JunB. These two fusion proteins were expressed in bacteria and bound to glutathione beads. In comparison to the amino terminus of c-Jun, neither the amino terminus of c-Jun with serines 63 and 73 mutated to leucine (Fig. 2A, lane 1) nor the amino terminus of JunB (Fig.  2 A , lane 2) could function as substrates for this purified PK ( Fig. 2 A , compare lanes 1 and 2 with 3 ) . This result suggests that the enzyme which we have purified functions similarly in vitro to the PK which is activated by phorbol ester treatment of U937 cells phosphorylating the amino terminus of c-Jun on serines 63 and 73 but not the amino terminus of JunB.
Because phorbol ester treatment of U937 cells activates PKC, we examined whether the c-Jun amino-terminal PK was activated by serinelthreonine phosphorylation and whether dephosphorylated c-Jun kinase could be reactivated by PKC. To examine whether the c-Jun PK could be inactivated by dephosphorylation, it was incubated with phosphatase 2A for 30 (Fig. 3, lane 3 ) or 60 min (Fig. 3, lane 4 ) at 37 "C. This treatment completely inactivates the cJAT-PK (Fig. 3, compare lanes 3 and 4 with 1 and 2), suggesting that it is activated by serinelthreonine phosphorylation. However, it can be reactivated after phosphatase treatment, by PKC stimulated with calcium and phospholipids (Fig. 3, compare lanes 3 and 4 with 5 and 6) . However, PKC alone is not capable of stimulating the phosphorylation of the GST:c-Jun peptide (Fig. 3, lane 9). Also, a pseudosubstrate inhibitor of PKC (21), while not affecting cJAT-PK directly (Fig. 3, lane  2), inhibited the ability of the PKC with and without calcium and phospholipids to reactivate the cJAT-PK (Fig. 3, compare lanes 5 and 6 with 7 and 8). These results demonstrate that PKC mediated phosphorylation of the cJAT-PK.
The cJAT-PK is capable of phosphorylating c-Jun but not A B 1 2 3 FIG. 2. A, cJAT-PK does not phosphorylate c-Jun with serines 63 and 73 mutated to leucines or the amino terminus of Jun B. cJAT-PK was incubated with beads containing equivalent amounts of each fusion proteins; the reaction was terminated after 10 min. In lane I , GST:c-Jun containing serines mutated to leucines was used as a substrate, while in lane 2, GSTamino-terminal 89 amino acids of JunB, and in lane 3, GSTc-Jun was used as a substrate. B, cJAT-PK is capable of phosphorylating the entire c-Jun molecule. c-Jun was purified from bacterial extract and used as a substrate for the c-JAT PK in a 10-min PK assay. An autoradiogram of a 10% SDS gel is shown. The 46-kDa molecular mass marker and the phosphorylated c-Jun are denoted by arrows.  (15, 16)) play a role in mediating the binding of c-JAT PK to the substrate, we have evaluated the ability of increasing equivalent amounts of GSTv-Jun amino-terminal fusion protein or GST:c-Jun amino terminus containing serines 63 and 73 mutated to leucines to bind the cJAT-PK. The enzyme was incubated with the beads containing either fusion protein, and the beads were then spun out. The superntant was assayed for GSTc-Jun PK activity (see "Experimental Procedures"). As the amount of GST:v-Jun beads used in the preincubation was increased from 2 to 16 pg there was a 60% decrease (Fig.  4, lanes 2-5) in the amount of c-JAT PK activity assayed in the supernatant. However, when the enzyme was incubated with an identical concentration of beads bound with GSTc-Jun containing serine to leucine mutations at positions 63 and 73 (GST:c-Jun mut) at all concentrations tested, the beads bound greater than 95% of the kinase activity (Fig. 4,  lanes 7-10). At the smallest amount of beads used in the preincubation (Fig. 4, compare lanes 10, c-Jun mut and 2, v-Jun), GSTc-Jun mut bound 50-fold more enzyme activity than the equivalent v-Jun beads. Similar results were obtained with GSTc-Jun beads. Beads containing only the GST fusion did not bind this PK activity (Fig. 4, lane I ) and if GST:c-Jun mut beads were used as a substrate no phoshorylation occurred (Fig. 4, lane 6). This experiment suggests that the 27-amino acid NHp-terminal amino acids found in c-Jun and not v-Jun functions to mediate the binding of the cJAT-PK.
Previous results have demonstrated that serines 63 and 73 can be phosphorylated by pp42/44 and pp54 MAP kinases in uitro (7). Using a partially purified preparation of these kinases, we find that they are capable of phosphorylating the GST:c-Jun fusion protein (data not shown). These kinases can be inactivated by phosphatase 2A, but they cannot be reactivated by PKC to phosphorylate GST:c-Jun fusion (data not shown). However, the material eluted from the heparin-Sepharose and the GSTc-Jun affinity column does not contain pp42,44 enzymes as demonstrated by Western bloting using a monoclonal antibody specific for pp42,44 MAP kinase. We have also found that the M07e human leukemic cell line, when treated with phorbol esters has little cJAT-PK activity but contains equivalent amounts of activated pp42 MAP kinase to that seen in U937 cells (data not shown). This suggests that pp42 is not the cJAT-PK. The molecular weight of the protein kinase eluted from SDS gels is considerably different from the pp54 MAP kinase. However, the pp54 has been purified from rat, and the human kinase could be somewhat larger. Although the cJAT-PK is also capable of phosphorylating MAP-2 (data not shown), it is not one of the MAP kinases previously described to phosphorylate c-Jun. Since higher molecular weight MAP-2 phosphorylating enzymes have been discovered, it is possible that this cJAT-PK is related to the MAP family of enzymes (18).
Our results suggest that cJAT-PK can be inactivated by serine/threonine dephosphorylation and reactivated by PKC. This suggests that there is a PK cascade leading to c-Jun phosphorylation. The existence of such a cascade would be strengthened by the demonstration both in uitro and in uiuo of PKC phosphorylation of the c-JAT protein kinase. Previous in uiuo results from our laboratory have suggested that serine/threonine dephosphosphorylation may play a role in regulating the c-Jun PK. Treatment of U937 cells with okadaic acid, a serine phosphatase inhibitor, increases the phosphorylation of c-Jun protein and enhances the phosphorylation stimulated by PKC activators (8).
The fact that the 27-amino acid domain deleted in v-Jun but present in c-Jun appears to play an important role in both transformation and transcription mediated by the c-Jun protein. This region has been shown to bind a potential inhibitor of transcriptional activation (15). In addition, mutations within this domain increase the transforming activity of c-dun (17). Our data suggest that the absence of this domain greatly inhibits the binding of the cJAT-PK to the c-Jun protein. Could the c-JAT PK be either a transcriptional inhibitory protein or a regulator of the transcriptional inhibitor by phosphorylating serines 63 and 73? Further experiments will be necessary to examine these possibilities.