(Received for publication, October 17, 1994; and in revised form, December 15, 1994)
From the
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.
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 ()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.
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).
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.
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 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.
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 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.
Figure 5:
Molecular weight estimation of the
UV-stimulated p53 kinase by gel filtration chromatography on Superose 6
(10/30). 2 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.
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 -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).
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.
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.