©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
p53 Is Phosphorylated in Vitro and in Vivo by an Ultraviolet Radiation-induced Protein Kinase Characteristic of the c-Jun Kinase, JNK1 (*)

(Received for publication, October 17, 1994; and in revised form, December 15, 1994)

Diane M. Milne Linda E. Campbell David G. Campbell (1) David W. Meek (§)

From the Biomedical Research Center, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, United Kingdom and the Medical Research Council Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee DD1 4HN, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The p53 tumor suppressor protein is thought to play a major role in the defense of the cell against agents that damage DNA. In this report, we describe the identification and characterization of a protein kinase that phosphorylates mouse p53 at a single site, serine 34, a major site of phosphorylation in the cell. The protein kinase is activated strikingly following treatment of cells with ultraviolet radiation, has a native molecular weight of approximately 45,000, and can be resolved from mitogen-activated protein (MAP) kinase by chromatography on Superose 6 and DEAE-cellulose. The p53 kinase activity co-purifies with UV-activated c-Jun kinase activity on heparin-Sepharose and on a c-Jun (but not a v-Jun-) affinity column. Treatment of the partially purified kinase with CL100, a protein phosphatase that specifically dephosphorylates MAP kinase homologues, inhibits its activity. Taken together, the data suggest that this p53 kinase is likely to be activated by phosphorylation and may be a member of the stress-activated protein kinase subfamily of MAP kinases. UV irradiation of SV3T3 cells leads to increased phosphorylation of p53 at serine 34, indicating that phosphorylation of p53 by this kinase is likely to be physiological. Phosphorylation of p53 by this protein kinase may be a key event in a signal transduction mechanism that coordinately controls key nuclear proteins in response to oxidative stress or DNA damaging agents.


INTRODUCTION

The p53 tumor suppressor protein (recently reviewed by Donehower and Bradley(1) ) is a nuclear phosphoprotein that is activated in response to a variety of DNA-damaging agents(2, 3) . Activation of p53 leads to cell growth arrest at the G1/S boundary (3, 4) or the induction of apoptosis(5, 6) , thereby preventing the proliferation of genetically damaged cells(7) . Loss of p53 suppressor function through mutation is a common event in the development of a wide variety of human cancers (8) and may contribute to an increase in the number of genetic abnormalities(9, 10) . p53 nullizygous mice develop normally but become susceptible to spontaneous tumor formation at an early age (11) , supporting the view that p53 functions principally as a tumor suppressor. At the molecular level, p53 potently transactivates promoters containing a p53-responsive DNA sequence element (12, 13, 14, 15, 16, 17, 18) but represses other promoters (19, 20) through its interaction with the TATA-binding protein(21, 22, 23, 24) . In particular, p53-dependent induction of p21, an inhibitor of cyclin-dependent kinases(25) , may mediate the biological effects of p53 (at least in part) (26, 27, 28) by blocking phosphorylation-dependent inactivation of the retinoblastoma protein(29, 30, 31) .

p53 is multiply phosphorylated at amino- and carboxyl-terminal sites in vivo(32, 33, 34, 35) and by several different protein kinases in vitro including p34cdc2(36, 37) , double-stranded DNA-activated protein kinase(34, 38) , casein kinase I(39) , casein kinase II(40, 41) , and MAP (^1)kinase(42) . Phosphorylation of the casein kinase II site (serine 386 in mouse p53) activates the growth suppressor activity of p53(43) . Similarly, phosphorylation of p53 by casein kinase II in vitro potently activates its specific DNA binding activity(44) . Phosphorylation of p53 by double-stranded DNA-activated protein kinase is also thought to contribute to p53-dependent cell cycle arrest(45) . The effects of phosphorylation of p53 by other protein kinases are less well defined.

The cellular response to DNA damage involves a number of events including an increase in the activities of several protein kinases (reviewed by Anderson(46) ). Among these, a novel c-Jun kinase termed JNK1 (which is a member of the stress-activated protein kinase family (47) ) is activated following UV irradiation of cells(48, 49) . We have previously presented evidence that mouse p53 is phosphorylated by members of the MAP kinase family in response to serum and also UV radiation(42) . In this paper, we show that following UV irradiation of cells, a second p53 kinase, which we have identified as JNK1 (or a highly related kinase), is activated in addition to MAP kinase. This second kinase phosphorylates mouse p53 at serine 34, a major site of phosphorylation in the cell, and its activation is temporally distinct from the activation of MAP kinase. The data suggest that phosphorylation of p53 by UV- or stress-activated protein kinases may be important in the response of cells to DNA-damaging agents.


EXPERIMENTAL PROCEDURES

Cell Lines

C57MG cells (50) were a gift from C. Dickson (Imperial Cancer Research Fund Laboratories). C57MG cells and simian virus 40-transformed NIH3T3 cells (SV3T3) were routinely used and were maintained in Dulbecco-Vogt's modified Eagle's medium supplemented with 10% fetal bovine serum.

Construction and Expression of Glutathione S-Transferase-p53 and Glutathione S-Transferase-c-Jun Fusion Proteins

Recombinant glutathione S-transferase-p53 (GST-p53) fusion proteins, expressed using the vector pGEX-2T (Amrad Corp. Ltd.), were used in this study. The following GST-p53 fusions were constructed, and their salient features are summarized in Fig. 1. To express fusion protein 221 (FP221), a 250-base pair NcoI fragment encoding amino acids 1-85 of wild type mouse p53 was purified, and the single-stranded ends were filled in using DNA polymerase I. The fragment was then cloned in the correct reading frame and sense orientation with respect to the GST gene into the SmaI site of pGEX-2T. FP221 contains potential phosphorylation sites for casein kinase I, DNA-activated protein kinase, MAP kinase (ERK2), and the serine 34 protein kinase(51) . As a control, the NcoI fragment was cloned into pGEX-2T in the opposite orientation to express FP222. To construct FP267, the FP221 plasmid was cut with EcoRI and partially with PstI (cutting at p53 codon 65). The single-stranded ends were removed using mung bean nuclease, and the plasmid was recircularized. This fusion encodes a protein that terminates at amino acid 65 of the p53 and therefore lacks the MAP kinase sites (threonines 73 and 83(42) ). To construct the FP279 plasmid, the 154-base pair SacI fragment encoding amino acids 11-63 of mouse p53 was cloned into the M13 vector mp18. The p53 sequences were then taken back out of mp18 using BamHI and EcoRI and transferred into pGEX-2T to give the correct reading frame and sense orientation with respect to the GST gene. FP274 was generated by changing the codon for serine 34 in the FP221 plasmid to an alanine codon using an Amersham oligonucleotide-directed mutagenesis kit. Similarly, FP294 is a derivative of FP279 in which the serine 34 codon has been mutated to encode alanine.


Figure 1: Nomenclature and salient features of glutathione S-transferase-p53 fusion proteins. The glutathione S-transferase-p53 fusion protein FP221 is shown diagramatically. The amino acid sequences of mouse p53 from amino acids 21-38 and 56-85 are given. The darkareas represent p53 amino acid sequences from 1-20 and 39-55 while the large open circle represents the glutathione S-transferase component of the fusion protein. Trypsin cleavage sites are shown, and phosphorylation sites are indicated by the letter P within an opencircle. The kinases that phosphorylate these sites are as follows: CK1, casein kinase 1; DNA-PK, double-stranded DNA-activated protein kinase; and MAPK, mitogen-activated protein kinase. The salient features of the six GST-p53 fusion proteins used in this study are listed below the diagram.



The GST-c-Jun (FP299) fusion protein contains amino acids 1-257 of chicken c-Jun, while the GST-v-Jun protein has amino acids 1-230 of v-Jun. Both were a gift from Dr. D. Gillespie (Beatson Cancer Research Institute, Glasgow).

UV Irradiation of Cells and Preparation of Cell Lysates

Cells at 80-90% confluency were shifted into medium containing 0.5% calf serum for 48 h. The medium was removed, and the cells were rinsed three times in phosphate-buffered saline and then exposed to a 50 Jm dose of germicidal UV radiation using a Stratalinker (Stratagene). The medium was replaced, and the cells were incubated for various times up to 90 min. Extracts were prepared by washing the cells three times in ice-cold phosphate-buffered saline and lysed in 1 ml/plate of ice-cold Nonidet P-40-buffer (10 mM sodium phosphate, pH 7.2, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 1 mM NaF, and 1 mM benzamidine). Lysates were cleared by centrifugation at 20,000 times g for 1 h at 4 °C. Lysates were aliquoted, frozen in liquid nitrogen, and stored until required.

Radiolabeling of Cells

SV3T3 cells were seeded on 15-cm plates and were labeled (at 80% confluency) for 4 h with 5 mCi of [P]orthophosphoric acid in 15 ml of phosphate-free Dulbecco-Vogt's modified Eagle's medium containing 10% dialyzed fetal bovine serum. Cells were harvested as described above.

Immunoprecipitation, Precipitation of Fusion Proteins, and SDS-Polyacrylamide Gel Electrophoresis

The polyclonal serum CM5 was a gift from C. Midgley (CRC Cell Transformation Group, University of Dundee). Immunoprecipitation of full-length p53 was carried out as previously described(42) . Lysates of bacterial cells expressing GST-p53 fusion proteins were stored at -70 °C. Fusions proteins were isolated by incubation of the lysates with glutathione-Sepharose beads (Pharmacia Biotech Inc.) at 4 °C for 1 h with rocking. The beads were then washed twice in phosphate-buffered saline and once with 50 mM Tris, pH 7.5, before being used in kinase assays.

Protein samples were resolved by polyacrylamide gel electrophoresis in 10% gels (for full-length p53) or 12.5% gels (for the fusion protein) in the presence of 0.1% SDS. Autoradiography was carried out at -70 °C with Fuji RX film and intensifying screens.

Affinity Purification of the c-Jun Kinase, JNK1

c-Jun kinase (JNK1) was prepared as described by Hibi et al.(48) . Briefly, lysates prepared from UV-treated cells were rocked at 4 °C for 3 h with 10 µg of GST-c-Jun fusion protein immobilized onto 100 µl (wet volume) of reduced glutathione-Sepharose beads (Pharmacia). The beads were washed five times with 25 mM Tris, pH 7.5, 50 mM NaCl, 2.5 mM MgCl(2), 0.1 mM EDTA, and 0.05% Triton X-100. The Jun kinase was eluted by incubating the beads for 10 min at 4 °C in the wash buffer containing 2 M urea. The beads were pelleted by centrifugation, and the supernatants were dialyzed overnight at 4 °C into 25 mM Tris, pH 7.5, 10 mM MgCl(2), 0.1 mM EGTA, and 10% (v/v) glycerol.

Western Blotting

Proteins were separated on a SDS 10% polyacrylamide gel and transferred to nylon filters (Immobilon, Pharmacia). The blots were probed with ER16, an anti-ERK-specific monoclonal antibody (Signal Transduction Laboratories Inc.), and detected by chemiluminescence using an ECL kit (Amersham Corp.) according to the manufacturer's instructions.

Protein Kinase Assays

p53 kinase assays were carried out as previously described (42) using GST-p53 or GST-c-Jun fusion proteins immobilized on Sepharose beads as substrates. The phosphorylated proteins were resolved by SDS-polyacrylamide gel electrophoresis and detected by autoradiography as described above. Phosphorylated p53 was quantitated either as previously described (40) or by using a GS-250 Molecular Imager (Bio-Rad).

Two-dimensional Phosphopeptide Analysis and Phosphoamino Acid Analysis

Two-dimensional phosphopeptide analysis was carried out as previously described(33, 52) . Phosphoamino acid analysis was also carried out as previously described(53) . Thin layer plates were exposed to pre-flashed Fuji RX film at -70 °C with intensifying screens. Phosphopeptide maps were quantitated using a GS-250 Molecular Imager (Bio-Rad).

Edman Degradation of Phosphorylated p53

Phosphorylated p53 was digested with trypsin as previously described(33) , and the phosphopeptides were purified by reverse phase chromatography on HPLC. The phosphorylated peptides were covalently coupled to a polyvinylidene difluoride disc using a Sequelon-AA kit (Millipore) as described by the manufacturer. The peptides were sequenced using an Applied Biosystems 470A gas phase protein sequencer. The amino acids released at any given cycle were eluted by 90% methanol without loss of protein from the filter. Quantitation of P released was carried out after each round of the Edman cycle by liquid scintillation counting.


RESULTS

p53 Is Phosphorylated in Vitro by Two Distinct Protein Kinases That Are Activated at Different Times Following UV Irradiation of C57MG Cells

We have previously presented evidence that two sites in the amino-terminal region of mouse p53 (threonines 73 and 83) are phosphorylated in vitro by the MAP kinases p42 and p44 following stimulation of C57MG cells by serum or UV irradiation(42) . To determine whether additional p53-directed protein kinase activities were activated in response to UV irradiation, quiescent C57MG cells were treated with 50 Jm of bacteriocidal UV radiation; the cells were harvested at various times following irradiation, and lysates were prepared and used as a source of p53 kinase activity. The ability of these lysates to phosphorylate a GST-p53 fusion protein (FP221) containing the amino-terminal 85 amino acids of mouse p53 was examined; this and other fusion proteins employed in this study are presented in Fig. 1. As a control, the cells were stimulated by the addition of fetal bovine serum to a final concentration of 20% (v/v). We have already established that both of these stimuli activate MAP kinases under the conditions employed here (42) .

The data (Fig. 2A) show that there was already a p53 kinase activity present in the unstimulated cells. Following UV treatment, a substantial increase in p53 kinase activity occurred within 10 min and persisted until the 90-min time point. This kinase activity peaked at 10-20 min post-irradiation and decayed slowly over the time course. Closer examination of the data in Fig. 2A revealed that two closely migrating bands corresponding to phosphorylated FP221 were discernible, especially at the 10- and 20-min time points. Strikingly, the lower of these two bands (which was absent at the 0-min time point) peaked at 10 min, while the upper band did not reach a maximum value until 20-30 min post-irradiation. Myelin basic protein kinase activity peaked at 10 min (data not shown), suggesting that the lower band might arise from phosphorylation of FP221 by MAP kinase. Kinase activities were also present in the lysates of cells treated with 20% serum for 5 min, but in this case the lower band was strikingly predominant (Fig. 2A, laneSER), even at later times after serum stimulation (data not shown). To control for the possibility that the kinases were phosphorylating GST sequences and not the p53 domain, the kinase reactions were carried out using a control fusion protein (FP222, Fig. 1) expressed from a plasmid in which the amino-terminal 85 codons of p53 were fused to the GST gene in the antisense orientation (Fig. 2C). Using this fusion protein, no significant GST kinase activity could be detected, even after prolonged exposure. The data therefore indicate that UV radiation induces two protein kinases that peak at different times after irradiation and that phosphorylate p53 at different amino-terminal sites.


Figure 2: Phosphorylation of GST-p53 fusion proteins by untreated and UV-stimulated C57MG cell lysates. 10 times 5-cm plates of 70% confluent C57MG cells were shifted into medium containing 0.5% calf serum for 48 h and then irradiated with 50 Jm of germicidal UV radiation as described under ``Experimental Procedures.'' After replacing the medium, the cells were harvested at 0, 5, 10, 20, 30, 60, and 90 min post-irradiation. As a control, one plate of cells was treated with 20% fetal bovine serum in place of UV irradiation and was harvested 7 min after the addition of the serum (SER). Cell lysates were prepared, and protein kinase activities were carried out as described under ``Experimental Procedures'' using 5 µl of lysate as the source of enzyme. The exposure time for autoradiography for the gels in panelsA and B was 15 h, while the exposure time for panelC was 2 days.



Since we had previously shown that MAP kinases are activated under these conditions(42) , the kinase assays were carried out using a p53 substrate that lacked the MAP kinase phosphorylation sites (FP267; Fig. 1). The data once again indicated that a p53 kinase was induced by UV treatment of the cells, but in this case the peak of activity clearly occurred at 20-30 min post-irradiation, and there was no indication of any differential mobility shifting on the gel. The data therefore suggest that there is a UV-induced protein kinase, distinct from the p42 and p44 MAP kinases, that can phosphorylate mouse p53 within its amino-terminal 63 amino acids.

The UV-stimulated Protein Kinase Phosphorylates Mouse p53 at Serine 34, Which Is a Major Physiological Site of Phosphorylation

To identify the site at which p53 was phosphorylated by this novel protein kinase, fusion protein FP267 was phosphorylated using a C57MG cell lysate, which had been irradiated with 50 Jm UV 20 min prior to cell lysis. Phosphoamino acid analysis of the phosphorylated protein revealed that only phosphoserine was present (data not shown). The phosphorylated protein was then digested with trypsin, and the phosphopeptides were purified by reverse phase chromatography on the HPLC, using a 0-100% acetonitrile gradient. One major phosphopeptide was observed, eluting at about 24% acetonitrile, together with several minor phosphopeptides (data not shown; the FP267 protein also contains phosphorylation sites for casein kinase I and the double-stranded DNA-activated protein kinase(51) ). Radiosequence analysis of this major phosphopeptide showed the release of radioactive material at cycle 10 of the Edman degradation (Fig. 3). Taken together with the phosphoamino acid analysis and the predicted cleavage sites for trypsin within the p53 domain (Fig. 1), these data indicated that the UV-stimulated kinase phosphorylated serine 34, which is a major site of phosphorylation of mouse p53 in the cell(33, 34) .


Figure 3: Radiosequence analysis of the GST-p53 fusion protein FP221 phosphorylated by a UV-stimulated C57MG cell lysate. FP267 (which lacks the MAP kinase phosphorylation sites) was phosphorylated in vitro by a C57MG cell lysate that had been prepared 20 min following treatment of the cells with 50 Jm of UV radiation. The phosphorylation conditions were as described under ``Experimental Procedures'' except that 10 µM ATP at a specific activity of 100 Ci/mmol was used. The phosphorylated protein was then digested with trypsin, and the peptides were resolved by high performance liquid chromatography on a reverse phase C18 column. The major peak of radioactive material eluting from the column (at 24% acetonitrile) was subjected to automated protein sequencing. The radioactivity released at each cycle of the Edman degradation was determined by liquid scintillation counting.



To confirm that serine 34 was the residue targeted by the UV-stimulated kinase, the codon for serine 34 was changed to alanine by oligonucleotide-directed mutagenesis and substituted into the FP221 plasmid. FP221 and the new fusion protein, FP274 (Fig. 1), were then phosphorylated in vitro using extracts from untreated and UV-irradiated cells, after which the proteins were analyzed by tryptic phosphopeptide mapping. The data (Fig. 4) showed that there were no differences in the phosphopeptide patterns of the mutant fusion protein FP274 whether phosphorylated with the unstimulated cell lysate or the lysate from the cells prepared at 20 min post-irradiation (compare panelsC and D). However, when fusion protein FP221 (containing the wild type p53 amino-terminal 85 amino acids) was used as substrate, a novel major phosphopeptide appeared only after phosphorylation by the lysate from the UV-treated cells (compare panelsA and B). This new phosphopeptide is very hydrophobic and migrates to the position characteristic of the phosphoserine 34-containing tryptic peptide (see Fig. 9). Taken together, these data clearly identify serine 34 as the target of the UV-stimulated protein kinase.


Figure 4: Two-dimensional tryptic phosphopeptide analysis of the GST-p53 fusion protein FP221 phosphorylated by unstimulated and UV-stimulated C57MG cell lysates. Fusions proteins FP221 and FP274 (containing the alanine 34 mutation) were phosphorylated using extracts of C57MG cells that had been untreated or irradiated with 50 Jm of UV radiation and then harvested after 20 min. The phosphorylated proteins were resolved by SDS-polyacrylamide gel electrophoresis, eluted from the gel, and prepared for two-dimensional tryptic phosphopeptide mapping as described under ``Experimental Procedures.'' Phosphopeptides were separated in the horizontal direction by electrophoresis at pH 1.9 and in the vertical direction by chromatography. The samples were as follows: A and C, FP221 and FP274, respectively, each phosphorylated by the lysate from untreated cells; B and D, FP221 and FP274, respectively, each phosphorylated by the lysate from UV-irradiated cells. The origins are marked with arrowheads, and the serine 34 peptide is indicated in panelB by an arrow.




Figure 9: Phosphorylation of p53 at serine 34 is stimulated in SV3T3 cells following UV irradiation. 2 times 15-cm plates of 80% confluent SV3T3 cells were placed in medium containing 0.1% serum for 24 h and then labeled for 4 h with 5 mCi of [P]orthophosphate per plate as described under ``Experimental Procedures.'' Prior to harvesting the cells, one plate was exposed to 50 Jm of UV radiation. The other plate was left untreated as control. The cells were incubated for a further 30 min, after which they were harvested. The p53 proteins immunopreciptated and analyzed by two-dimensional tryptic phosphopeptide mapping as described under ``Experimental Procedures.'' The panels are as follows: A, control cells; B, UV-irradiated cells; C, the fusion protein FP279 phosphorylated by the UV-stimulated protein kinase in vitro; and D, a mix of the phosphopeptides from panelsA and C.



The UV-stimulated Protein Kinase Has a Molecular Weight of Approximately 45,000 and Can Be Resolved from the MAP Kinase pp42by Chromatography on Superose 6

To characterize the UV-stimulated kinase, C57MG cells were placed in 0.1% serum for 48 h and then irradiated with 50 Jm of UV radiation followed by incubation in medium for a further 20 min. Untreated cells were used as controls. Lysates prepared from the UV-treated and untreated cells were mixed with marker proteins of known molecular weight and fractionated by fast protein liquid chromatography using a Superose 6 (gel filtration) column. The fractions were assayed for p53 kinase activity using the fusion protein FP221 as substrate. The data show that there were low levels of p53 kinase activity present in the fractionated lysates of the untreated cells (Fig. 5A). However, 20 min following UV irradiation, a single major peak of p53 kinase activity was detected (Fig. 5B). When compared with the standard markers, the molecular weight of the UV-activated protein kinase was estimated to be 45,000. Western analysis of the Superose 6 fractions, using the anti-MAP kinase antibody ER16 (Signal Transduction Laboratories Inc.) as probe, showed that the peak of p53 kinase activity eluted before the p42 isoform of MAP kinase (p42; Fig. 5C). The UV-stimulated p53 (serine 34) kinase could also be resolved from p42 on DEAE-cellulose (data not shown), confirming that the UV-stimulated kinase was not the p42 isoform of MAP kinase.


Figure 5: Molecular weight estimation of the UV-stimulated p53 kinase by gel filtration chromatography on Superose 6 (10/30). 2 times 15-cm plates of 80% confluent C57MG cells were shifted into medium containing 0.1% calf serum for 48 h. One plate was then exposed to 50 Jm of UV radiation, after which the cells were incubated for a further 20 min. As a control, the other plate was left untreated. Cell lysates were prepared, mixed with proteins of known molecular weight, and loaded onto a Superose 6 (10/30) gel filtration column attached to the fast protein liquid chromatography and equilibrated in 50 mM Tris, pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 10 mM 2-mercaptoethanol, and 5% (v/v) glycerol. 10 ml of buffer were passed through the column before collection of 0.5-ml fractions was initiated. The fractions were assayed for p53 kinase and c-Jun kinase activity; the p53 substrate proteins were FP221 (panelsA and B), FP279 (D), and FP294 (E), and the Jun substrate was the c-Jun fusion protein FP299 (F). Fractions were analyzed by Western blotting for MAP kinase using the monoclonal antibody ER16 (Signal Transduction Laboratories Inc.). The molecular weight markers (with their respective abbreviations and native molecular weights given in parentheses) were yeast alcohol dehydrogenase (ADH, 150,000), bovine serum albumin (BSA, 66,000), ovalbumin (OV, 45,000), carbonic anhydrase (CA, 29,000), and lysozyme (LYS, 14,000). The fractions containing the peak amounts of these proteins were identified by staining the gel with Coomassie Blue, and their elution positions are indicated by arrows.



The fusion protein FP221 has phosphorylation sites for other kinases in addition to serine 34 (Fig. 1). The fractions were therefore assayed against the fusion protein FP279 (Fig. 1) in which the CK1 and MAP kinase phosphorylation sites are absent. Once again, a single major peak of p53 kinase activity was observed (Fig. 5D). When the same fractions were assayed against the fusion protein FP294 (which is the alanine 34 version of FP279 (Fig. 1)), no p53 kinase activity could be detected (Fig. 5E), indicating that the UV-activated kinase was targeting serine 34. Recently, JNK1, a novel UV-activated protein kinase of molecular weight 46,000, was identified and shown to phosphorylate serines 63 and 73 at the amino terminus of the c-Jun protein(48, 49) . The Superose 6 fractions were therefore assayed against FP299, a GST-c-Jun fusion protein containing the amino-terminal 257 amino acids of c-Jun. The data (Fig. 5F) showed that the p53 kinase and c-Jun kinase activities co-purified on Superose 6. The c-Jun kinase activity was not detectable in the control lysates (data not shown). These data therefore suggest that the p53 kinase and JNK1 might be the same kinase.

p53 Is Phosphorylated at Serine 34 by a UV-activated Protein Kinase Purified by Affinity Chromatography on c-Jun or Heparin Columns

To determine whether the UV-stimulated p53 kinase and JNK1 were the same kinase, control and UV-stimulated lysates were analyzed by chromatography on a c-Jun affinity column and on heparin-Sepharose. To prepare the c-Jun column, the c-Jun fusion protein FP299 was adsorbed onto glutathione-Sepharose beads, and the unbound proteins were washed away. The protein kinase was eluted from the c-Jun-Sepharose using a step of 2 M urea, followed by dialysis to remove the urea, as described by Hibi and co-workers(48) . The eluted protein was then assayed for c-Jun and p53 kinase activities, respectively. The data (Fig. 6) show that there was Jun kinase activity in the untreated cell lysate (panelA, lane1) and in the ``unbound protein'' fraction from the c-Jun beads, but none of this activity was retained by and eluted from the column (lane3). When the UV-stimulated cell lysate was used, there was again protein kinase activity in the lysate and unbound fractions (lanes4 and 5, respectively), but this time significant c-Jun kinase could be eluted from the column (lane6). A similar pattern of results was observed when p53 was used as a substrate (panelsB and C). No p53 kinase activity from the untreated cells was eluted from the c-Jun column (lanes3) (it should be noted that the lysates contain other p53 kinase activities, which phosphorylate different sites from serine 34). However, following fractionation of the UV-treated cell lysate, p53 kinase activity was clearly eluted from the c-Jun column (lanes6). It should be noted that some c-Jun kinase activity was also present in the p53 kinase assays (lanes6) and probably resulted from uncoupling of c-Jun-GST from the glutathione-Sepharose column in the presence of the urea. When the alanine 34 mutant of the p53 (FP294) was used as a substrate, no p53 kinase activity could be detected after elution from the column (panelD, lane6), confirming that the c-Jun-bound kinase phosphorylates serine 34 in p53. When a v-Jun-GST fusion protein was used as the affinity matrix, neither c-Jun kinase nor p53 kinase activities were immobilized by the column (data not shown) (JNK1 binds to, and can be eluted from, a c-Jun but not a v-Jun column(48) ). Taken together, these data strongly argue that the UV-stimulated p53 kinase is indeed JNK1 or a highly related kinase.


Figure 6: Phosphorylation of GST-p53 and GST-c-Jun fusion proteins by affinity-purified UV-stimulated c-Jun kinase. The affinity-purified kinase was prepared as described under ``Experimental Procedures'' and then assayed against the GST-c-Jun fusion protein (panelA) and the GST-p53 fusion proteins FP221 (panelB), FP279 (panelC), and FP294 (panelD). The samples were as follows: 1-3, untreated cell lysate; 4-6, UV-irradiated cell lysates. 1 and 4, lysates before purification; 2 and 5, kinase activity remaining in lysate after incubation with immobilized c-Jun; 3 and 6, kinase activities eluted from the immobilized Jun by 2 M urea.



Jun kinase activity also binds tightly to heparin(54) . Lysates from UV-irradiated cells were therefore adsorbed to heparin-Sepharose (heparin HiTrap, Pharmacia) and were eluted using a linear gradient of NaCl (Fig. 7). The UV-stimulated p53 kinase activity eluted as a single peak at 700 mM NaCl (panelA), while c-Jun kinase activity eluted as two closely migrating peaks, the first of which co-eluted with p53 kinase activity (panelB). When v-Jun was used in the kinase assays in place of c-Jun, only the second peak appeared, indicating that only the first peak of kinase activity targeted serines 63 and 73 (v-Jun is not phosphorylated at these amino-terminal sites(55) ). Once again, the co-purification of p53 kinase and c-Jun amino-terminal kinase supports the idea that these two protein kinase activities are the same.


Figure 7: Analysis of p53 kinase activities following fractionation of UV-irradiated C57MG cell lysates on heparin-Sepharose. UV-stimulated cell lysate was prepared as described under ``Experimental Procedures.'' The proteins were resolved by fast protein liquid chromatography using a heparin-Sepharose (HiTrap, Pharmacia) column (bed volume, 5 ml) equilibrated in 50 mM Tris (pH 7.5) containing 10 mM beta-mercaptoethanol and 1 mM benzamidine. The column was washed with this buffer until the A of the material flowing through the column reached a minimum value. The proteins were eluted with a linear gradient of increasing sodium chloride concentration in the same buffer. 1-ml fractions were collected. A, elution profile of p53-serine 34 kinase activity, using fusion protein FP279 as substrate (closedcircles); B, elution profile of Jun kinase activity, using GST-Jun fusion proteins (the opencircles show c-Jun kinase activity while the closedsquares show v-Jun kinase activity).



The UV-stimulated Protein Kinase Can Be Inactivated by Treatment with CL100, a Threonine-Tyrosine Dual Specificity Protein Kinase

JNK1 is a member of the stress-activated protein (SAP) kinase subfamily of MAP kinases and contains a Thr-Pro-Tyr motif within kinase subdomain VIII(47) . Within this motif, both the threonine and the tyrosine residues must be phosphorylated for JNK1 to be active(49) . CL100 is a stress- and mitogen-inducible protein phosphatase(56) . Recently, it has been shown that CL100 is a dual specificity threonine-tyrosine phosphatase that specifically dephosphorylates and inactivates MAP kinase(57) . At least in vitro, CL100 inactivates several MAP kinase family members including ERKs 1 and 2(57) , the yeast MAP kinase FUS3, (^2)and a recently characterized MAP kinase isoform responsible for activating MAPKAP kinase 2(58) . We have therefore used CL100 to determine if the p53 kinase is a member of the MAP kinase family. In this experiment, the UV-stimulated p53 kinase was partially purified by gel filtration (Fig. 5) and then incubated for various lengths of time in the presence of homogeneous recombinant CL100. The kinase was then assayed for its ability to phosphorylate both c-Jun or p53 as described above. In control incubations, 1 mM sodium orthovanadate was included to inhibit the activity of CL100. The data (Fig. 8) show that, in the presence of 1 µg/ml CL100, both c-Jun kinase (panelA) and p53 kinase (panelB) activities were inactivated with similar kinetics over the course of 60 min (lanes3-6). This inactivation was inhibited when 1 mM sodium orthovanadate was included in the reaction (lanes 7-10). Vanadate itself had no effect on the activity of the protein kinase (compare lanes1 (control) with lanes2 (1 mM sodium orthovanadate)). These data were reproducible in three separate experiments and suggest that the UV-responsive kinase is activated by a dual phosphorylation event and is a MAP kinase-like enzyme.


Figure 8: Inactivation of UV-induced p53 kinase and c-Jun kinase activity by the dual specificity protein phosphatase CL100. 20-µl aliquots of the peak fraction of UV-induced p53 kinase activity from the Superose 6 column (Fig. 5) were incubated with 1 µg of homogeneous recombinant CL100 phosphatase for up to 60 min. The reactions were stopped by the addition of sodium orthovanadate to a final concentration of 1 mM. In control incubations, sodium orthovanadate was added to a final concentration of 1 mM at the same time as the CL100 was added. The phosphatase-treated kinase was then assayed for its ability to phosphorylate the GST-c-Jun fusion protein FP299 (panelA) or the GST-p53 fusion protein FP279 (panelB). Protein kinase assays were carried out as described under ``Experimental Procedures.'' The samples were as follows: 1, UV-stimulated kinase with no additions; 2, UV-induced kinase incubated in the presence of 1 mM sodium orthovanadate for 60 min; 3-6, UV-induced kinase incubated in the presence of 1 µg of CL100 for 10, 20, 30, and 60 min, respectively; 7-10, UV-induced kinase incubated in the presence of 1 µg of CL100 and 1 mM sodium orthovanadate for 10, 20, 30, and 60 min, respectively.



UV Irradiation of SV3T3 Cells Leads to Increased Phosphorylation of p53 at Serine 34

To determine whether irradiation of cells leads to phosphorylation of p53 at serine 34, SV3T3 cells were labeled with [P]orthophosphate for 4 h. The cells were then exposed to 50 Jm of UV radiation and were harvested 30 min after this treatment. As a control, an identical plate of labeled cells was not irradiated (SV3T3 cells were used because they contain very high levels of p53, which provides sufficient material for carrying out tryptic phosphopeptide mapping (42) ). Following UV irradiation, an increase in the intensity of only one phosphopeptide (marked with an arrow in Fig. 9, panelB) was observed. Quantitative analysis of this peptide in the control versus UV-treated maps (measured relative to the intensity of the other phosphopeptides in each map) indicated that the phosphorylation was increased by 65%. To determine whether this peptide corresponded to serine 34, the fusion protein FP279 was phosphorylated in vitro by the UV-stimulated kinase. Following digestion with trypsin, the phosphopeptides from this reaction were separated alone (panelC) or were mixed, before two-dimensional separation, with phosphopeptides from p53, which had been labeled in the SV3T3 cells. The data showed that the phosphopeptide from FP279 co-migrated with the p53 peptide, whose phosphorylation was increased in response to UV. Taken together, these data provide strong evidence that p53 is phosphorylated at serine 34 in vivo in response to UV radiation.


DISCUSSION

In this paper, we report the identification and characterization of a novel p53 kinase that is potently activated following UV irradiation of cells. The kinase phosphorylates mouse p53 at a single residue, serine 34 ( Fig. 3and Fig. 4). Serine 34 has been previously reported to be a site that is phosphorylated in vivo(33, 34) , and our data have confirmed this observation (Fig. 9). Moreover, UV irradiation leads to an increase in the phosphorylation of p53 in the cell, specifically at serine 34, indicating that p53 is likely to be a physiological target of this kinase (Fig. 9). To maximize any effect in looking for changes in p53 phosphorylation after UV irradiation, we have carried out the analysis at a time when the kinase activity (in vitro) appears to reach a maximum value (i.e. 30 min post-irradiation) (Fig. 2). The effect of UV on stimulating phosphorylation of p53 at this site is modest. However, we note that the UV-activated kinase is already detectable in SV3T3 cell lysates before UV treatment (data not shown) and that serine 34 is already phosphorylated in the SV3T3 cells (Fig. 9A). This may explain why the increase in phosphorylation at serine 34 after UV treatment is small. Moreover, it has been recently reported that p53 is activated in response to stress induced by radioisotopic labeling(59) , and it is therefore possible that the phosphorylation state of p53 may already have been changed simply by virtue of setting up the experiment. Nevertheless, the increase in phosphorylation of p53 at serine 34 following UV irradiation is striking and suggests that p53 is targeted in response to cellular stress through a phosphorylation mechanism. Clearly, development of some non-radioactive method for identifying changes in phosphorylation at specific sites will be useful in probing further the factors that govern phosphorylation of p53. We are currently exploring this idea.

Recently, a novel c-Jun kinase termed JNK1 was identified in cells that had been irradiated by UV(48, 49) . JNK1 was also recently shown to be a member of the stress-activated protein kinase subfamily of MAP kinases, which are activated by a number of agents including UV, heat shock, and cycloheximide(47) . Our data strongly suggest that the UV-activated p53 kinase is indeed JNK1 (or a closely related kinase) based on the following observations: 1) both the c-Jun and p53 kinase activities are stimulated by UV irradiation of cells (Fig. 5) in a dose-dependent manner (data not shown); 2) both activities co-purify on several different types of purification media (Fig. 5, Fig. 6, and Fig. 8); 3) strikingly, both the p53 (serine 34) and c-Jun kinase activities co-purify on an affinity column composed of the c-Jun protein (Fig. 6) but not the v-Jun protein (data not shown; JNK1 associates tightly with c-Jun, but not v-Jun(48) ); and 4) both activities can be inactivated by the tyrosine-threonine dual specificity protein phosphatase CL100, which shows a high degree of specificity in its interaction with substrates (57) (this is the first demonstration that CL100 is active toward a member of the SAP kinases). In addition, we have shown conclusively that the UV-activated p53 kinase is not MAP kinase (p42) based on the observations that 1) the UV-activated kinase can be resolved from p42 by gel filtration (Fig. 5) or ion exchange chromatography (data not shown); 2) the UV-activated kinase phosphorylates a different site in mouse p53 from MAP kinase (Fig. 3)(42) ; 3) following UV irradiation of cells, the activity of the UV-activated kinase peaks at a later time and is sustained longer than MAP kinase (Fig. 2); and 4) the mobility shift of the p53 substrate induced after phosphorylation by the UV-activated kinase is different from that which arises following phosphorylation by MAP kinase (Fig. 2).

The mechanism of activation of JNK1 by bacteriocidal UV (UVC) is still unclear. This mechanism appears not to be initiated through DNA damage but through events at the cell membrane, possibly involving oxidative stress(60) . It is therefore not clear at present what physiological agents induce the phosphorylation of p53 at serine 34. However, the identification of p53 as an additional physiological substrate for a SAP kinase suggests that there may well be several nuclear targets for these kinases and that the involvement of p53 may be part of a coordinated response to cellular insult. It is also unclear how phosphorylation of p53 by SAP kinases may control p53 function. Analysis of the phosphorylation of p53 is complex because of its multi-site nature. Understanding the role of individual phosphorylation events will require analysis at both the cellular level (for example, by studying the behavior of phosphorylation site mutants in mammalian cells) and at the biochemical level where p53 activity can be measured in vitro following phosphorylation by highly purified kinases. Future studies of this type should reveal the potential regulatory role of serine 34 phosphorylation in p53 function.


FOOTNOTES

*
This work was supported by the Medical Research Council (UK). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The recipient of a Medical Research Council Senior Non-clinical Fellowship. To whom correspondence should be addressed. Tel.: 44-382-660111 ext. 3517 (office) or 3514 (laboratory); Fax: 44-382-69993.

(^1)
The abbreviations used are: MAP, mitogen-activated protein; GST-p53, glutathione S-transferase-p53; HPLC, high performance liquid chromatography; SAP, stress-activated protein; FP, fusion protein.

(^2)
S. M. Keyes and A. Gartner, unpublished data.


ACKNOWLEDGEMENTS

We thank Philip Cohen, Frances Fuller-Pace, and Steve Keyse for critically reviewing the manuscript. We are also grateful to David Gillespie (Beatson Cancer Research Institute, Glasgow) for the gift of vectors expressing GST-Jun fusion proteins and to Steve Keyse (Biomedical Research Center, Dundee) for the gift of purified recombinant CL100 phosphatase.


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