Vol. 189, No. 3, 1992 December 30, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1701-l 708
PHOSPHORYLATION-ASSOCIATED CONFORMATION SHIFT OF ANTI-ONCOGENE PHOSPHOPROTEIN D53 IN CONCANAVALIN-A ACTIVATED HUMAN T LYMPHOCYTES
John E. McClure,
Jonathan
A. McClure,
and William
T. Shearer
Departments of Pediatrics, M icrobiology, Immunology, and Molecular Virology, Baylor College of Medicine, Allergy-Immunology Service, Texas Children's Hospital, Houston, TX 77030 Received November 18, 1992 an anti-peptide A new radioimmunoassay for ~53, employing conserved Domain V, exhibited antibody directed against specificity for a relatively dephosphorylated form of ~53. This form, correlated with the monoclonal antibody PAB421+ conformation, appeared transiently in the cytosol of cycloheximide-treated T cells undergoing activation by concanavalin-A/serum. Concurrently, there were decreased levels of p53 in the nucleus that correlated with increased phosphorylation of ~53. After 90 m in nuclear levels of p53 increased steadily above levels of unstimulated cells. Anti-p53 antibodies introduced into cells prior to stimulation enhanced cell proliferation in response to m itogens. 0 1192Academic Pxe*s,Inc.
Progress in understanding the role of anti-oncogene p53 in cell cycle control has been hampered by the lack of sensitive assays for the protein found in normal cells at low levels. Synthetic peptides representing conserved Domain V of p53 were used for preparation of anti-peptide antiserum S372 and for competition RIA of ~53. Domain V was selected for study since the primary amino acid sequence contains hydrophilic residues and :i s part of the molecule responsible for binding of SV-40 Large T antigen. Domain V, therefore, m ight be externally disposed in native p53 and be capable of binding S372 in competition RIA. The DNA sequence coding for conserved Domain V lies immediately upstream of a major identified nuclear localization signal(l) and PBS, phosphate-buffered saline, FBS, fetal bovine serum, Tyr, tyrosine, Cys, cysteine, RIA, radioinununoassay, BSA, Con-A, concanavalinbovine serum albumin, MAB,monoclonal antibody, A, SDS-PAGE,sodium dodecylsulfate-polyacrylamide gel electrophoresis, NLS, nuclear localization signal sequence. Abbreviations:
1701
0006-291 X/92 $4.00 Copjjright 0 1992 b?; Academic Press, Inc. All rights of reproduction in an\ ,form reserved.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 189, No. 3, 1992
has
proximity
to
two
serine
residues
(315
and
392)
that
are
phosphorylated by the cell cycle-associated kinases ~34~~~~' kinase and casein kinase II, respectively (2,3). Several authors(4,5,6) have proposed that the extent of p53 phosphorylation determines its conformation, sub-cellular localization, and biological M ilner (7) observed in Con-A-stimulated T lymphocytes a function. shift of p53 conformation from the resting state(PAB246+,PAB421-) to the activated state(PAB246-,PAB421+). Enhanced p53 reactivity with PAB421 correlates with low levels of phosphorylation and cell proliferation while low PAB421 reactivity correlates with high levels of phosphorylation and with anti-proliferation (8,9). reacted poorly in We had observed that rp53 from sf9 cells RIA compared with its reaction with S372 on Western blots. Here we found that recognition of rp53 by 5372 was phosphorylation dependent since digestion of sf9 protein extracts or nuclear extracts of T cells produced increased amounts of IRp53 in the RIA. The RIA, having an apparent specificity for a relatively dephosphorylated form of ~53, was utilized in studies of IRp53 levels in sub-cellular compartments of T cells undergoing stimulation with Con-A/serum. Previously we had observed (10) a pattern of change in IRp53 levels during lymphocyte activation identical to that described here when we utilized a two-site MABbased RIA for p53 employing PAB421 as reporter antibody. MATERIALS AND METHODS Preparation of peptide antiserum.
synthetic
~53
Domain
V
peptides
and
anti-
Two peptides representing human p53 Domain V synthesized having the sequences (residues 270-286) were The amino (C/Y)FEVRVCACPGRDRRTEE as previously reported (11). acid compositions and concentration of final purified products (Cys-p53V and Tyr-p53V) were verified by amino acid analysis. Cysp53V was conjugated to BSA and used for antibody production in and Tyr-p53V was used for preparation of 12'1-Tyr-p53V rabbits, tracer and for assay standard. Specific anti-p53 antibody (S372) was detected by its ability to precipitate 1251-tyr-p53V and by Western blot reactivity against protein extracts of T and B lymphocytes and of sf9 cells producing rp53. Protocol for the radioimmunoassay. A complete description of the RIA is in preparation (McClure and Shearer,unpublished). Briefly, the peptide Tyr-p53V was radioiodinated to high specific activity(150-200pCi/pgJ by means of Na1251(IMS-300, Amersham Corp., 400-500 mCi/ml) and chloramine-T using the method of Greenwood et al. (12). The product "'I-Tyr-p53V was purified using a column of Bio-gel P-2 (Bio-Rad Lab.). Into 12X75mm glass tubes were delivered 0.9ml of antibody S372(1:80,000) diluted in assay buffer (5OmM sodium citrate pH 6.5, 1501nM sodium chloride, and 0.1% gelatin) and l-100@ of either peptide standard or protein Following incubation for 60 m in at 37', 1251-Tyr-p53V extract. 1702
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(30,000 cpm/tube) was added and incubated an additional 60 min. Separation of bound from free tracer was accomplished by addition and radioactivity in protein of carrier IgG and second antibody, in a gamma counter. The least precipitates was determined and the within-assay coefficient detectable dose was =20 pg/tube, analysis was performed using of variance was 11%. Statistical Student's t-test. alkaline phosphatase recombinant and Preparation of I?53 digestion. Cloned human p53 was introduced into the baculovirus Autouraoha californica nuclear polyhedrosis virus (AcMNPV),which was used for infections of insect Swodowte a frualwerda (sf9) Maxwell (Univ. of Texis Cancer Center, cells by Dr. S. Houston,TX) . Cells infected with either wild-type AcMNPV or p53recombinant virus were lysed, and protein extracts of cytosol and nucleus were prepared by a modification of the method of Dignam et al. (12). The presence of rp53 was confirmed by SDS-PAGE and Western blot using PAB421 S372. For ti vitro deand phosphorylation experiments,protein extracts (150pg/ml) were digested with calf intestinal alkaline phosphatase(300 units/ml, Calbiochem, LaJolla,CA) plus staurosporin(lp, Calbiochem) for 2-4 hr at 37' and then assayed for IRp53 by RIA. Isolation of T preparation of protein lymphocytes and extracts. T lymphocytes were isolated from donor buffy coat concentrates by first selectively removing B cells by means of magnetic particles coated with anti-CD19 antibody(Pan-B Dynabeads, Dynal,AS, Oslo, Norway). Following density centrifugation over a layer of Ficoll (LSM, Organon Teknika, Durham, NC) and culture in RPMI-1640 without FBS overnight, the non-adherent cells, enriched for T cells were recovered for stimulation experiments. In a typical experiment, 30 X lo6 cells/tube were stimulated, lysed, and extracted as described(l3). The cytosolic proteins were recovered in Buffer A (1OmM HEPES pH 7.9, 1.5mM MgC12, 1OmM KCl, 0.5mM DTT, and lpg/ml each of protease inhibitors pepstatin, aprotinin, and leupeptin), and nuclear proteins were extracted in Buffer C(2OmM HEPES pH 7.9, 25% glycerol, 0.42M sodium chloride, 1.51t-M MgCl;!, 0.2mM EDTA). Activation of con-A. All T cells were T lymphocytes with suspended in RPM1 plus cycloheximide(25pg/ml) for 60 min. Cells exposed to agents in addition to Con-A,either staurosporin(lw) or phosphatase inhibitors(20mM sodium fluoride, 20mM sodium pyrophosphate, and 0.4mM sodium ortho-vanadate) were pre-treated for 15 min prior to cell stimulation. The cells were aliquoted in 1.5ml microcentrifuge tubes(30 X lo6 cells/ml), the stimulants ConA(lOpg/ml) and 10% FBS were added, and at intervals the cells were Cell disruption pelleted by centrifugation at 750Xg for 2 min. were carried out as and preparation of protein extracts described(l2). Equivalent amounts of protein were assayed for p53 content by RIA. Intracellular loading of antibody and cell proliferation. T lymphocytes (20 X lo6 cells/0.5ml) were suspended in solutions of The suspensions antibodies (lOOC(g/ml) to be loaded into cells. fields (Baekon 2000, were subjected to high intensity electric Baekon,Inc., Saratoga,CA). By using 1251-IgG we had determined that significant IgG(=l X lo6 molecules/cell) could be introduced into Following viable lymphocytes (McClure and Shearer, unpublished). electroporation, the cells were washed and dispensed into 96-well and activated by addition of microtiter plates(2 X lo5 cells/well), The extent of incorporation Of Con-A(2.5pg/ml) plus 10% FBS. lpCi/ml) into cellular DNA was [3H] thymidine(TRA.120, Amersham, measured at 24, 48, and 72hr. 1703
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DISCUSSION
The anti-peptide antibody S372 directed against Domain V of human p53 recognized an epitope displayed on native p53 since the The method of protein extraction did not employ detergents. specificity of S372 was examined by Western blot as shown in (1OOpg) of the proteins Fig.1. Lane 2 shows cytosolic lymphoblastoid cell line Raji and Lanes 3 and 4 show detection of The reaction p53 in the extract by PAB421 and S372, respectively. of S372 antibody with rp53 in a cytosolic extract(50pg) of sf9 of rp53 from Since some preparations cells is shown in Lane 5. sf9 cells had reacted weakly with S372 in the RIA but strongly we examined the effect of dewith 5372 in a Western blot, phosphorylation of p53 by alkaline phosphatase digestion, as shown in Fig.2. While there was no detectable IRp53 in a cytosolic protein extract of uninfected sf9 cells, there was a detectable infected level of IRp53 (98 * 11 pg/l5pg protein) in sf9 cells Upon digestion with alkaline with the recombinant baculovirus. more than fourphosphatase for 4hr, the level of IRp53 increased
600
01
Time (hr.) Fig.1. Specificity of anti-peptide antibody for ~53 by Western blot. An extract of cytosolic proteins of lymphoblastoid cell RajiClOO~g) was analyzed by lO%SDS-PAGE(Lane 2).Western blots of this extract were probed with MAB PAB421 (Lane 3) or S372(1:200 Lane 4) for detection of ~53. Lane 5 shows reaction between S372 infected and rp53 in cytosolic extract(50Fg protein) of sf9 cells with p53+-recombinant baculovirus.
Fig. 2. Increased immunoreactivity of ~53 toward S372 in RIA by de-phosphorylation using h vitro digestion with alkaline ghosphatase. Protein extracts (150pg/ml) were digested with alkaline phosphatase (300 units/ml) at 37O and digests were analyzed for IRp53 by RIA. Immunoreactivity increased 3-4 fold in 4 hr of digestion in extracts of sf9 cells and of T cell nuclear Similar results were obtained in three experiments. proteins. 1704
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280 260 240
100 oUnsUmulsted Control OwnA SUmdated AStaurwporln + co”A APhosphstase Inhibiters
80 60
220 200 + conA
160 160
40
140 120 100
0
0 0
20
40
60
80
100
120
140
160
160
0
200
20
40
60
80
Time p.s. (min.)
100
120
140
160
180
Time p.s. (min.)
Fig. 3. Sub-cellular levels of IRg53 in T cells stimulated with Con-A/serum. IRp53 was detectable in cytosol immediately upon cell stimulation and this response was diminished 50% by staurosporin and almost totally inhibited by phosphatase inhibitors. Nuclear IRp53 levels declined, presumably due to enhanced pi3 phosphorylation in response to mitogen, and then rose above control levels at ,90 min. Staurosporin partially inhibited while phosphatase inhibitors enhanced the level of nuclear phosphorylation of ~53. This result was repeated in three trials.
fold(to
480
f
39
pg/15pg
In
protein).
IRp53 in an extract of nuclear proteins from 95 f 11 to 300 k 31 pg/l5/.lg protein. that
5372
recognized
controlled
by
the
conformation
level
of
as
protein's
epitope
an
Domain
important
sub-cellular
presented
of
level
from T cells These data V access
of phosphorylation probably determined
p53,
phosphorylation,
of
~53. by its
feature
localization
in
and
of
increased suggested
to which A model
was for
degree
ofi
regulating
function
the
has
been
(14,15). Upon
A(lOpg/ml) cytosol
exposure plus
at
protein min
an
the
addition,
serum
levels
(Fig.3A).
protein
the
were near control control levels[at 21 pg/2Opg
protein
for
only
nucleus
30 min
k
T cells
IRp53
a maximum
was of
At
14
to
nuclear
10 min IRp53
f
IRp53
=177 levels
10
were
f
12
in
the by 90 were
declined pg/2Opg
increasing,
and then increased at 90 min, control =235 k 16 and stimulated Staurosporin attenuated the (p=O.OOl)].
levels 180 min
Con-
pg/SOpg
detection of IRp53
nuclear
stimulated
to
detected 108
to below limits of decreased levels
(Fig.3B). =254
By
5 min,
reached
and declined Concurrently,
control
(p=O.O02).
cycloheximide-treated
that
by 20 min
observed in by 30%,from
of
above =277 k changes
in IRp53 in both cell compartments by 50%. Decreased levels of indicate that IRp53 in the cytosol readily-soluble may protein (16) is a anchor phosphorylation of cytoplasmic prerequisite to p53 release or that a protein phosphatase that acts upon ~53 itself must be phosphorylated before a conformation 1705
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it is shift in p53 can occur. Within the nuclear compartment,since likely that the decreases in IRp53 levels are indicative of the effect of staurosporin in increased ~53 phosphorylation, attenuating the decrease in IRp53 m ight be explained as inhibition p53(2). of a kinase such as p34 cdc-2 known to phosphorylate Phosphatase inhibitors almost completely abrogated the appearance of IRp53 in the cytosol and accentuated the decrease in the nuclear IRp53 levels or enhanced levels of phosphorylated p53 [nuclear IRp53 at 10 m in,control =148 & 9 and stimulated =177f13 pg/2Opg protein(p=0.05)]. Protein phosphatases in the cytoplasm may be critical to p53 mobilization for nuclear translocation in a manner similar to the transcription factor SW15 of S.cerevisiae (17) and the eukaryotic transcription factor NF-AT (18). These factors translocated to the nucleus only when they had been acted upon by cytoplasmic protein phosphatases. The effect of phosphatase inhibitors on accentuating the decrease in nuclear IRp53 observed in our study probably reflects an accumulation of phosphorylated p53 in the nucleus during the first 30 m in in After 90 m in in the presence of response to m itogenic signal. phosphatase inhibitors there were no increases in nuclear IRp53 above control levels since increased IRp53 was dependent upon phosphatase activity. These following data suggest the conclusions: m itogenic stimulation promoted the release of p53 from cytoplasmic anchor protein(s) and its dephosphorylation exposing the NLS as well as Domain V epitope. Once p53 had it was phosphorylated in order to translocated to the nucleus, nuclear function(s). Finally, the relatively carry out its dephosphorylated form of p53 (PAB42l+conformer) began to accumulate as the cells began to move toward S phase of the cell cycle. If p53 is tightly sequestered by cytoplasmic anchor protein(s) in resting cells (16) and becomes more readily soluble during cell activation, introduction of anti-p53 antibodies may have an effect on the proliferative response to con-A/serum stimulation. Control IgG or anti-p53 antibodies, PAB122 or affinity-purified S372, were loaded into T cells that were subsequently stimulated with Con-A/serum.In contrast to results of Mercer et al.(19) who m icro-injected anti-p53 MAB into nuclei of fibroblasts and observed inhibition of cell proliferation in m itogenic stimulation, our response to measurements of proliferation of the electroporated cells (Table 1) indicated that having anti-p53 antibodies present in the cells at the time of 1706
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Table 1. Effect of anti-p53 antibodies electroporated into T cells subsequently stimulated with Con-A/serum was enhanced proliferation.Following electroporation of antibody, equivalent numbers (2 X 105) of viable cells were dispensed into 96-well microtiter plates and stimulated with addition of Con-A(2Spg/ml) and lO%FBS. DNA synthesis was measured by addition of [sH]thymidine (lpCi/ml) for the final 12 hr of culture. These data are renrcsentative of three indenendent exoeriments. DNA Synthesis [-‘H]Thymidine incorporated / 2 X 105 cells ( cpm
+ S.E.M. )
24 hr
Treatment No Electroporation - Con-A
2Y5+
+ Con-A Electroporation + Con-A Non-immune IgG
48 hr 58
479+
72 hr 71
1,069 +
146
27,533, 2211
64,978* 2838
95,457 + 13,182
5,482f 667
a 43,034& 1796
47,645 rt 1439
PAB 122
10,438 + 1471
S372
21,X48+1567
2497 *
b51,862f
73,874 III 3147
64,068 + 2908
87,531 31 7714
$ t-test. b vs. a (p=O.O3). All other comparisons (p=O.OOl).
stimulation
enhanced
Translocation was important mitogenic role
p53
results
as
demonstrate
and cell
in
studies cycle
consistent
the
of RIA,
the
role
of
by the
p53
with
cell shift
in
in
72hr.
antibodies,
response the
growth
capable
conformation of
50-90%
proliferative
regulator
that
phosphorylation-dependent useful
inhibited
nucleus, the
an observation a negative
by
proliferation
of p53 to the for attenuating
stimulus,
of
cell
of of
intracellular
to
the
recognized (20).
These
detecting ~53,
will
a be
signalling
control.
ACKNOWLEDGMENTS
This work was supported by the Immunology Research Fund of the Texas Children's Hospital. The authors would like to thank Dr. Abraham Kuruvilla for many helpful discussions. Dr. Janet Butel provided the hybridoma cell lines PAB421 and PAB122 and, along with Dr. Christine Noonan, gave many helpful suggestions. Dr. Richard Cook performed peptide synthesis and purification, and Dr. Steven Maxwell provided sf9 cells expressing recombinant ~53.
REFERENCES
1. Addison,C., Jenkins,J.R., and Stiirzbecher,H.W.(1990), Oncogene, 5, 423-426. 2. Stiirzbecher,H.W., Maimets,T., Chumakov,P., Brain,R.,Addison,C. Simanis,V., Rudge,K., Philps,R., Grimaldi,M., Court,W., and Jenkins,J.R. (1990) Oncogene, 5, 795-801. 3. Tack,L.C. and Wright,J.H. (1992) J.Virology, 66, 1312-1320. 4. Milner,J. (1991) Proc.Roy.Soc.Lond., B, 139-145. 1707
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AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5. Ullrich,S.J., Anderson,C.W., Mercer,W.E., and Appella,E.(l992) J.Biol.Chem. 267, 15259-15262. 6. Suzuki,K., Ono,T., and Takahashi,K. (1992) Bioc.Biophys. Res.Comm. 183, 1175-1183. 7. M ilner,J. (1984) Nature, 310, 143-145. 8. Ullrich,S.J., Mercer,W.E., and Appella,E. (1992) Oncogene 7, 1635-1643. 9. M ilner,J. and Watson,J.V. (1990) Oncogene, 5, 1683-1690. 10. McClure,J.E. and Shearer,W.T. (1990) FASEB J., 3, 2420a. 11. Buchman,V.L.,Chumakov,P.,Ninkina,N.,Samarina,O.,and Georgiev,G. (1988) Gene, 70, 245-252. 12. Greenwood,F.C., Hunter,W.M., and Glover,J.S. (1963) Biochem.J., 89, 114-121. 13. Dignam,J.D., Lebovitz,R.M., and Roeder,R.G. (1983) Nucl.Acids Res., 11, 1475-1482. 14. M ilner,J. and Medcalf,E.A. (1991) Cell 65, 765-774. 15. Zerrahn,J., Deppert,W., Weidemann,D., Patchinsky,J., Richards,F., and M ilner,J. (1992) Oncogene 7, 1371-1381. 16. Gannon,J.V. and Lane,D.P. (1991) Nature, 349, 802-806. 17. Moll,J.v., Tebb,G., Surana,U., Robitsch,H., and Nasmyth,K., (1991) Cell 66, 743-758. 18. Flanagan,W.F., Corthesy,B., Bram,R.J., and Crabtree,G.R., (1991) Nature, 352, 803-807. 19. Mercer,W.E., Nelson,D., DeLeo,A.B., Old,L.J., and Baserga,R. (1982) Proc.Nat.Acad.Sci.(USA), 79, 6309-6312. 20. Finlay,C., Hinds,P.W., and Levine,A.J. (1989) Cell, 57, 1083-1093.
1708
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1701-l 708
PHOSPHORYLATION-ASSOCIATED CONFORMATION SHIFT OF ANTI-ONCOGENE PHOSPHOPROTEIN D53 IN CONCANAVALIN-A ACTIVATED HUMAN T LYMPHOCYTES
John E. McClure,
Jonathan
A. McClure,
and William
T. Shearer
Departments of Pediatrics, M icrobiology, Immunology, and Molecular Virology, Baylor College of Medicine, Allergy-Immunology Service, Texas Children's Hospital, Houston, TX 77030 Received November 18, 1992 an anti-peptide A new radioimmunoassay for ~53, employing conserved Domain V, exhibited antibody directed against specificity for a relatively dephosphorylated form of ~53. This form, correlated with the monoclonal antibody PAB421+ conformation, appeared transiently in the cytosol of cycloheximide-treated T cells undergoing activation by concanavalin-A/serum. Concurrently, there were decreased levels of p53 in the nucleus that correlated with increased phosphorylation of ~53. After 90 m in nuclear levels of p53 increased steadily above levels of unstimulated cells. Anti-p53 antibodies introduced into cells prior to stimulation enhanced cell proliferation in response to m itogens. 0 1192Academic Pxe*s,Inc.
Progress in understanding the role of anti-oncogene p53 in cell cycle control has been hampered by the lack of sensitive assays for the protein found in normal cells at low levels. Synthetic peptides representing conserved Domain V of p53 were used for preparation of anti-peptide antiserum S372 and for competition RIA of ~53. Domain V was selected for study since the primary amino acid sequence contains hydrophilic residues and :i s part of the molecule responsible for binding of SV-40 Large T antigen. Domain V, therefore, m ight be externally disposed in native p53 and be capable of binding S372 in competition RIA. The DNA sequence coding for conserved Domain V lies immediately upstream of a major identified nuclear localization signal(l) and PBS, phosphate-buffered saline, FBS, fetal bovine serum, Tyr, tyrosine, Cys, cysteine, RIA, radioinununoassay, BSA, Con-A, concanavalinbovine serum albumin, MAB,monoclonal antibody, A, SDS-PAGE,sodium dodecylsulfate-polyacrylamide gel electrophoresis, NLS, nuclear localization signal sequence. Abbreviations:
1701
0006-291 X/92 $4.00 Copjjright 0 1992 b?; Academic Press, Inc. All rights of reproduction in an\ ,form reserved.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 189, No. 3, 1992
has
proximity
to
two
serine
residues
(315
and
392)
that
are
phosphorylated by the cell cycle-associated kinases ~34~~~~' kinase and casein kinase II, respectively (2,3). Several authors(4,5,6) have proposed that the extent of p53 phosphorylation determines its conformation, sub-cellular localization, and biological M ilner (7) observed in Con-A-stimulated T lymphocytes a function. shift of p53 conformation from the resting state(PAB246+,PAB421-) to the activated state(PAB246-,PAB421+). Enhanced p53 reactivity with PAB421 correlates with low levels of phosphorylation and cell proliferation while low PAB421 reactivity correlates with high levels of phosphorylation and with anti-proliferation (8,9). reacted poorly in We had observed that rp53 from sf9 cells RIA compared with its reaction with S372 on Western blots. Here we found that recognition of rp53 by 5372 was phosphorylation dependent since digestion of sf9 protein extracts or nuclear extracts of T cells produced increased amounts of IRp53 in the RIA. The RIA, having an apparent specificity for a relatively dephosphorylated form of ~53, was utilized in studies of IRp53 levels in sub-cellular compartments of T cells undergoing stimulation with Con-A/serum. Previously we had observed (10) a pattern of change in IRp53 levels during lymphocyte activation identical to that described here when we utilized a two-site MABbased RIA for p53 employing PAB421 as reporter antibody. MATERIALS AND METHODS Preparation of peptide antiserum.
synthetic
~53
Domain
V
peptides
and
anti-
Two peptides representing human p53 Domain V synthesized having the sequences (residues 270-286) were The amino (C/Y)FEVRVCACPGRDRRTEE as previously reported (11). acid compositions and concentration of final purified products (Cys-p53V and Tyr-p53V) were verified by amino acid analysis. Cysp53V was conjugated to BSA and used for antibody production in and Tyr-p53V was used for preparation of 12'1-Tyr-p53V rabbits, tracer and for assay standard. Specific anti-p53 antibody (S372) was detected by its ability to precipitate 1251-tyr-p53V and by Western blot reactivity against protein extracts of T and B lymphocytes and of sf9 cells producing rp53. Protocol for the radioimmunoassay. A complete description of the RIA is in preparation (McClure and Shearer,unpublished). Briefly, the peptide Tyr-p53V was radioiodinated to high specific activity(150-200pCi/pgJ by means of Na1251(IMS-300, Amersham Corp., 400-500 mCi/ml) and chloramine-T using the method of Greenwood et al. (12). The product "'I-Tyr-p53V was purified using a column of Bio-gel P-2 (Bio-Rad Lab.). Into 12X75mm glass tubes were delivered 0.9ml of antibody S372(1:80,000) diluted in assay buffer (5OmM sodium citrate pH 6.5, 1501nM sodium chloride, and 0.1% gelatin) and l-100@ of either peptide standard or protein Following incubation for 60 m in at 37', 1251-Tyr-p53V extract. 1702
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(30,000 cpm/tube) was added and incubated an additional 60 min. Separation of bound from free tracer was accomplished by addition and radioactivity in protein of carrier IgG and second antibody, in a gamma counter. The least precipitates was determined and the within-assay coefficient detectable dose was =20 pg/tube, analysis was performed using of variance was 11%. Statistical Student's t-test. alkaline phosphatase recombinant and Preparation of I?53 digestion. Cloned human p53 was introduced into the baculovirus Autouraoha californica nuclear polyhedrosis virus (AcMNPV),which was used for infections of insect Swodowte a frualwerda (sf9) Maxwell (Univ. of Texis Cancer Center, cells by Dr. S. Houston,TX) . Cells infected with either wild-type AcMNPV or p53recombinant virus were lysed, and protein extracts of cytosol and nucleus were prepared by a modification of the method of Dignam et al. (12). The presence of rp53 was confirmed by SDS-PAGE and Western blot using PAB421 S372. For ti vitro deand phosphorylation experiments,protein extracts (150pg/ml) were digested with calf intestinal alkaline phosphatase(300 units/ml, Calbiochem, LaJolla,CA) plus staurosporin(lp, Calbiochem) for 2-4 hr at 37' and then assayed for IRp53 by RIA. Isolation of T preparation of protein lymphocytes and extracts. T lymphocytes were isolated from donor buffy coat concentrates by first selectively removing B cells by means of magnetic particles coated with anti-CD19 antibody(Pan-B Dynabeads, Dynal,AS, Oslo, Norway). Following density centrifugation over a layer of Ficoll (LSM, Organon Teknika, Durham, NC) and culture in RPMI-1640 without FBS overnight, the non-adherent cells, enriched for T cells were recovered for stimulation experiments. In a typical experiment, 30 X lo6 cells/tube were stimulated, lysed, and extracted as described(l3). The cytosolic proteins were recovered in Buffer A (1OmM HEPES pH 7.9, 1.5mM MgC12, 1OmM KCl, 0.5mM DTT, and lpg/ml each of protease inhibitors pepstatin, aprotinin, and leupeptin), and nuclear proteins were extracted in Buffer C(2OmM HEPES pH 7.9, 25% glycerol, 0.42M sodium chloride, 1.51t-M MgCl;!, 0.2mM EDTA). Activation of con-A. All T cells were T lymphocytes with suspended in RPM1 plus cycloheximide(25pg/ml) for 60 min. Cells exposed to agents in addition to Con-A,either staurosporin(lw) or phosphatase inhibitors(20mM sodium fluoride, 20mM sodium pyrophosphate, and 0.4mM sodium ortho-vanadate) were pre-treated for 15 min prior to cell stimulation. The cells were aliquoted in 1.5ml microcentrifuge tubes(30 X lo6 cells/ml), the stimulants ConA(lOpg/ml) and 10% FBS were added, and at intervals the cells were Cell disruption pelleted by centrifugation at 750Xg for 2 min. were carried out as and preparation of protein extracts described(l2). Equivalent amounts of protein were assayed for p53 content by RIA. Intracellular loading of antibody and cell proliferation. T lymphocytes (20 X lo6 cells/0.5ml) were suspended in solutions of The suspensions antibodies (lOOC(g/ml) to be loaded into cells. fields (Baekon 2000, were subjected to high intensity electric Baekon,Inc., Saratoga,CA). By using 1251-IgG we had determined that significant IgG(=l X lo6 molecules/cell) could be introduced into Following viable lymphocytes (McClure and Shearer, unpublished). electroporation, the cells were washed and dispensed into 96-well and activated by addition of microtiter plates(2 X lo5 cells/well), The extent of incorporation Of Con-A(2.5pg/ml) plus 10% FBS. lpCi/ml) into cellular DNA was [3H] thymidine(TRA.120, Amersham, measured at 24, 48, and 72hr. 1703
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DISCUSSION
The anti-peptide antibody S372 directed against Domain V of human p53 recognized an epitope displayed on native p53 since the The method of protein extraction did not employ detergents. specificity of S372 was examined by Western blot as shown in (1OOpg) of the proteins Fig.1. Lane 2 shows cytosolic lymphoblastoid cell line Raji and Lanes 3 and 4 show detection of The reaction p53 in the extract by PAB421 and S372, respectively. of S372 antibody with rp53 in a cytosolic extract(50pg) of sf9 of rp53 from Since some preparations cells is shown in Lane 5. sf9 cells had reacted weakly with S372 in the RIA but strongly we examined the effect of dewith 5372 in a Western blot, phosphorylation of p53 by alkaline phosphatase digestion, as shown in Fig.2. While there was no detectable IRp53 in a cytosolic protein extract of uninfected sf9 cells, there was a detectable infected level of IRp53 (98 * 11 pg/l5pg protein) in sf9 cells Upon digestion with alkaline with the recombinant baculovirus. more than fourphosphatase for 4hr, the level of IRp53 increased
600
01
Time (hr.) Fig.1. Specificity of anti-peptide antibody for ~53 by Western blot. An extract of cytosolic proteins of lymphoblastoid cell RajiClOO~g) was analyzed by lO%SDS-PAGE(Lane 2).Western blots of this extract were probed with MAB PAB421 (Lane 3) or S372(1:200 Lane 4) for detection of ~53. Lane 5 shows reaction between S372 infected and rp53 in cytosolic extract(50Fg protein) of sf9 cells with p53+-recombinant baculovirus.
Fig. 2. Increased immunoreactivity of ~53 toward S372 in RIA by de-phosphorylation using h vitro digestion with alkaline ghosphatase. Protein extracts (150pg/ml) were digested with alkaline phosphatase (300 units/ml) at 37O and digests were analyzed for IRp53 by RIA. Immunoreactivity increased 3-4 fold in 4 hr of digestion in extracts of sf9 cells and of T cell nuclear Similar results were obtained in three experiments. proteins. 1704
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280 260 240
100 oUnsUmulsted Control OwnA SUmdated AStaurwporln + co”A APhosphstase Inhibiters
80 60
220 200 + conA
160 160
40
140 120 100
0
0 0
20
40
60
80
100
120
140
160
160
0
200
20
40
60
80
Time p.s. (min.)
100
120
140
160
180
Time p.s. (min.)
Fig. 3. Sub-cellular levels of IRg53 in T cells stimulated with Con-A/serum. IRp53 was detectable in cytosol immediately upon cell stimulation and this response was diminished 50% by staurosporin and almost totally inhibited by phosphatase inhibitors. Nuclear IRp53 levels declined, presumably due to enhanced pi3 phosphorylation in response to mitogen, and then rose above control levels at ,90 min. Staurosporin partially inhibited while phosphatase inhibitors enhanced the level of nuclear phosphorylation of ~53. This result was repeated in three trials.
fold(to
480
f
39
pg/15pg
In
protein).
IRp53 in an extract of nuclear proteins from 95 f 11 to 300 k 31 pg/l5/.lg protein. that
5372
recognized
controlled
by
the
conformation
level
of
as
protein's
epitope
an
Domain
important
sub-cellular
presented
of
level
from T cells These data V access
of phosphorylation probably determined
p53,
phosphorylation,
of
~53. by its
feature
localization
in
and
of
increased suggested
to which A model
was for
degree
ofi
regulating
function
the
has
been
(14,15). Upon
A(lOpg/ml) cytosol
exposure plus
at
protein min
an
the
addition,
serum
levels
(Fig.3A).
protein
the
were near control control levels[at 21 pg/2Opg
protein
for
only
nucleus
30 min
k
T cells
IRp53
a maximum
was of
At
14
to
nuclear
10 min IRp53
f
IRp53
=177 levels
10
were
f
12
in
the by 90 were
declined pg/2Opg
increasing,
and then increased at 90 min, control =235 k 16 and stimulated Staurosporin attenuated the (p=O.OOl)].
levels 180 min
Con-
pg/SOpg
detection of IRp53
nuclear
stimulated
to
detected 108
to below limits of decreased levels
(Fig.3B). =254
By
5 min,
reached
and declined Concurrently,
control
(p=O.O02).
cycloheximide-treated
that
by 20 min
observed in by 30%,from
of
above =277 k changes
in IRp53 in both cell compartments by 50%. Decreased levels of indicate that IRp53 in the cytosol readily-soluble may protein (16) is a anchor phosphorylation of cytoplasmic prerequisite to p53 release or that a protein phosphatase that acts upon ~53 itself must be phosphorylated before a conformation 1705
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it is shift in p53 can occur. Within the nuclear compartment,since likely that the decreases in IRp53 levels are indicative of the effect of staurosporin in increased ~53 phosphorylation, attenuating the decrease in IRp53 m ight be explained as inhibition p53(2). of a kinase such as p34 cdc-2 known to phosphorylate Phosphatase inhibitors almost completely abrogated the appearance of IRp53 in the cytosol and accentuated the decrease in the nuclear IRp53 levels or enhanced levels of phosphorylated p53 [nuclear IRp53 at 10 m in,control =148 & 9 and stimulated =177f13 pg/2Opg protein(p=0.05)]. Protein phosphatases in the cytoplasm may be critical to p53 mobilization for nuclear translocation in a manner similar to the transcription factor SW15 of S.cerevisiae (17) and the eukaryotic transcription factor NF-AT (18). These factors translocated to the nucleus only when they had been acted upon by cytoplasmic protein phosphatases. The effect of phosphatase inhibitors on accentuating the decrease in nuclear IRp53 observed in our study probably reflects an accumulation of phosphorylated p53 in the nucleus during the first 30 m in in After 90 m in in the presence of response to m itogenic signal. phosphatase inhibitors there were no increases in nuclear IRp53 above control levels since increased IRp53 was dependent upon phosphatase activity. These following data suggest the conclusions: m itogenic stimulation promoted the release of p53 from cytoplasmic anchor protein(s) and its dephosphorylation exposing the NLS as well as Domain V epitope. Once p53 had it was phosphorylated in order to translocated to the nucleus, nuclear function(s). Finally, the relatively carry out its dephosphorylated form of p53 (PAB42l+conformer) began to accumulate as the cells began to move toward S phase of the cell cycle. If p53 is tightly sequestered by cytoplasmic anchor protein(s) in resting cells (16) and becomes more readily soluble during cell activation, introduction of anti-p53 antibodies may have an effect on the proliferative response to con-A/serum stimulation. Control IgG or anti-p53 antibodies, PAB122 or affinity-purified S372, were loaded into T cells that were subsequently stimulated with Con-A/serum.In contrast to results of Mercer et al.(19) who m icro-injected anti-p53 MAB into nuclei of fibroblasts and observed inhibition of cell proliferation in m itogenic stimulation, our response to measurements of proliferation of the electroporated cells (Table 1) indicated that having anti-p53 antibodies present in the cells at the time of 1706
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Table 1. Effect of anti-p53 antibodies electroporated into T cells subsequently stimulated with Con-A/serum was enhanced proliferation.Following electroporation of antibody, equivalent numbers (2 X 105) of viable cells were dispensed into 96-well microtiter plates and stimulated with addition of Con-A(2Spg/ml) and lO%FBS. DNA synthesis was measured by addition of [sH]thymidine (lpCi/ml) for the final 12 hr of culture. These data are renrcsentative of three indenendent exoeriments. DNA Synthesis [-‘H]Thymidine incorporated / 2 X 105 cells ( cpm
+ S.E.M. )
24 hr
Treatment No Electroporation - Con-A
2Y5+
+ Con-A Electroporation + Con-A Non-immune IgG
48 hr 58
479+
72 hr 71
1,069 +
146
27,533, 2211
64,978* 2838
95,457 + 13,182
5,482f 667
a 43,034& 1796
47,645 rt 1439
PAB 122
10,438 + 1471
S372
21,X48+1567
2497 *
b51,862f
73,874 III 3147
64,068 + 2908
87,531 31 7714
$ t-test. b vs. a (p=O.O3). All other comparisons (p=O.OOl).
stimulation
enhanced
Translocation was important mitogenic role
p53
results
as
demonstrate
and cell
in
studies cycle
consistent
the
of RIA,
the
role
of
by the
p53
with
cell shift
in
in
72hr.
antibodies,
response the
growth
capable
conformation of
50-90%
proliferative
regulator
that
phosphorylation-dependent useful
inhibited
nucleus, the
an observation a negative
by
proliferation
of p53 to the for attenuating
stimulus,
of
cell
of of
intracellular
to
the
recognized (20).
These
detecting ~53,
will
a be
signalling
control.
ACKNOWLEDGMENTS
This work was supported by the Immunology Research Fund of the Texas Children's Hospital. The authors would like to thank Dr. Abraham Kuruvilla for many helpful discussions. Dr. Janet Butel provided the hybridoma cell lines PAB421 and PAB122 and, along with Dr. Christine Noonan, gave many helpful suggestions. Dr. Richard Cook performed peptide synthesis and purification, and Dr. Steven Maxwell provided sf9 cells expressing recombinant ~53.
REFERENCES
1. Addison,C., Jenkins,J.R., and Stiirzbecher,H.W.(1990), Oncogene, 5, 423-426. 2. Stiirzbecher,H.W., Maimets,T., Chumakov,P., Brain,R.,Addison,C. Simanis,V., Rudge,K., Philps,R., Grimaldi,M., Court,W., and Jenkins,J.R. (1990) Oncogene, 5, 795-801. 3. Tack,L.C. and Wright,J.H. (1992) J.Virology, 66, 1312-1320. 4. Milner,J. (1991) Proc.Roy.Soc.Lond., B, 139-145. 1707
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AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5. Ullrich,S.J., Anderson,C.W., Mercer,W.E., and Appella,E.(l992) J.Biol.Chem. 267, 15259-15262. 6. Suzuki,K., Ono,T., and Takahashi,K. (1992) Bioc.Biophys. Res.Comm. 183, 1175-1183. 7. M ilner,J. (1984) Nature, 310, 143-145. 8. Ullrich,S.J., Mercer,W.E., and Appella,E. (1992) Oncogene 7, 1635-1643. 9. M ilner,J. and Watson,J.V. (1990) Oncogene, 5, 1683-1690. 10. McClure,J.E. and Shearer,W.T. (1990) FASEB J., 3, 2420a. 11. Buchman,V.L.,Chumakov,P.,Ninkina,N.,Samarina,O.,and Georgiev,G. (1988) Gene, 70, 245-252. 12. Greenwood,F.C., Hunter,W.M., and Glover,J.S. (1963) Biochem.J., 89, 114-121. 13. Dignam,J.D., Lebovitz,R.M., and Roeder,R.G. (1983) Nucl.Acids Res., 11, 1475-1482. 14. M ilner,J. and Medcalf,E.A. (1991) Cell 65, 765-774. 15. Zerrahn,J., Deppert,W., Weidemann,D., Patchinsky,J., Richards,F., and M ilner,J. (1992) Oncogene 7, 1371-1381. 16. Gannon,J.V. and Lane,D.P. (1991) Nature, 349, 802-806. 17. Moll,J.v., Tebb,G., Surana,U., Robitsch,H., and Nasmyth,K., (1991) Cell 66, 743-758. 18. Flanagan,W.F., Corthesy,B., Bram,R.J., and Crabtree,G.R., (1991) Nature, 352, 803-807. 19. Mercer,W.E., Nelson,D., DeLeo,A.B., Old,L.J., and Baserga,R. (1982) Proc.Nat.Acad.Sci.(USA), 79, 6309-6312. 20. Finlay,C., Hinds,P.W., and Levine,A.J. (1989) Cell, 57, 1083-1093.
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