p53 protein:

guardian of
the genome
Review p53 protein function,
structure, and regulation, and
find tools and resources to
accelerate your p53 research.

1
Contents

Overview of p53 function and role in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

-- Cell cycle arrest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

-- DNA repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
-- p53 gene mutation and cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

p53 protein structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

MDM2 regulation of p53. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Post-translational modifications of p53. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

-- p53 phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
-- p53 acetylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

p53 antibody sampler panel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1
Overview of p53 function
and role in cancer

p53 is a tumor suppressor protein that regulates cell division and prevents tumor
formation by stopping cells with mutated or damaged DNA from dividing and signaling
for them to undergo apoptosis through transcriptional regulation.

Here we look at the function, structure, and modifications of p53 and outline the tools
you can use to accelerate your p53 research.

p53 is a transcription factor that activates a multitude of transcriptional targets in

response to cellular stress or DNA damage. A broad range of responses are coordinated
by p53 including cell cycle arrest, DNA repair, altered metabolism, anti-oxidant effects,
anti-angiogenic effects, autophagy, senescence and apoptosis (Bieging et al., 2014).

DNA damage Cellular stress

p53

Bax Gene regulation

Mitochondria Cell cycle arrest, cell

growth inhibition,
metabolic stress
Bcl-2 inhibition
Cyt-c

Apaf-1/Caspase 9 Apoptosis

Figure 1. Mechanisms of p53 function in apoptosis. . p53 responds to DNA damage or cellular stress and
activates the Bax and Apaf-1 pathways.

2
Cell cycle arrest

p53 can inhibit cell cycle progression in several ways. One way is through the
upregulation of p21 expression. p21 protein will then bind cyclin E/Cdk2 and cyclin D/
Cdk4 resulting in G1 arrest of the cell cycle (Wade-Harper et al., 1993). p53 can also bring
about cell cycle arrest at the G2/M phase through binding to other p53 target genes
such as 14-3-3σ (Martín-Caballero et al., 2001) and cdc25C (Clair et al., 2004).

DNA repair

p53 plays a role in DNA repair through both halting the cell cycle to allow repair
machinery to operate and directly through the activation of repair mechanisms
(Williams et al., 2016). p53 is commonly referred to as the “guardian of the genome”
because it constantly surveys the genome for signs of DNA damage such as
double-strand breaks. p53 is also known to play an active role in many different types
of DNA repair including nucleotide excision repair, base excision repair, mismatch
repair, and nonhomologous end-joining (Williams et al., 2016).

p53 gene mutation and cancer

TP53 (the gene encoding for p53) is the single most frequently mutated gene in human
cancer, with partial or complete loss of function occurring in over 50% of tumors
(Perri et al., 2016). Mutations in p53 confer a selective advantage on the tumor cells,
allowing them to evade cell cycle checkpoints, avoid apoptosis and senescence, and
proliferate under conditions where normal cells cannot (Pascual et al., 2019).

3
p53 protein structure

The p53 protein is active as a tetramer of 4 chains of 393 amino acids. Each chain has
several domains. At the N-terminal, there are two distinct transactivation domains (TADI
and TADII), a nuclear export signal (NES) followed by the proline-rich domain (PD) and
the DNA binding domain (DBD) (Wang et al., 1994).

The TADI (residues 1–42) and TADII (residues 43–62) are critical for p53 regulation as
they provide binding sites for the transcriptional machinery and the negative regulator
MDM2. The DBD (residues 102–292) is pivotal for the transcriptional activity of p53. It
contains 4 of the 5 conserved boxes in p53. The OD (residues 323–356) allows p53 to
form a tetramer which is organized as a dimer of dimers (Khoury et al., 2011).

At the C-terminus, there is an oligomerization domain (OD), three nuclear localization

signals (NLS), a second NES and a lysine-rich regulatory domain (RD). The cluster of
three NLSs mediate the nuclear location of the protein by binding to specific receptors
to allow selective passage of p53 through the nuclear pore complex.

The C-terminal NES, a highly conserved region has been shown to be essential for
nuclear export of p53. Both the NLS and NES regions are required for nuclear-cytosolic
shuttling of p53 to regulate p53 transcriptional function (Inoue et al., 2002).

N TADI TADII PD DBD OD RD C

1-42 43-62 63-97 102-292 323-356 363-393

Figure 2. p53 functional domains.

4
MDM2 regulation of p53

Murine double minute 2 (MDM2), is an E3 ubiquitin ligase responsible for the degradation
of p53 in wild type cells. p53 also induces the expression of MDM2, creating a p53/MDM2
feedback loop. Approximately 17% of tumors exhibit mdm2 gene amplification leading
to reduced levels and p53 and therefore poor prognosis for patient diagnosis. This makes
MDM2-p53 interaction a key target for cancer therapy (Nag et al., 2013).

Tools for MDM2 research:

MDM2
Antibody Anti-MDM2 [2A10] antibody

Anti-MDM2 (phospho S166) antibody

5
Post-translational
modifications of p53

When the cell is confronted with stress, p53 ubiquitylation is suppressed and p53
accumulates in the nucleus, where it is activated and stabilized by post-translational
modification including phosphorylation and acetylation (Dai et al., 2010).

Here we focus on some of these post-translational modifications (phosphorylation and

acetylation) and their role in p53 stress response.

p53 phosphorylation

Phosphorylation of p53 occurs rapidly in response to cellular stress. p53 contains multiple
serine and threonine residues that serve as phosphorylation sites for protein kinases
(Dai et al., 2010). These kinases include ATM/ATR, Chk1/Chk2, CK1, CK2, PKC, CDK1/2,
DNA-PK, HIPK2, ERK2, p38, and JNK.

CDK5 CHK2 CDK5 JNK CK1 CK1 CHK1 Aurora A GSK3ß CHK1 CK2
PRPK PLK3 DYRK2 CHK2 CDK2 PKC CHK2 PKR
AMPK HPK2 GSK3ß PKC CDK9
ARS ATM PKCδ p38
ATR p38 ARK5
ADNA-PK PRAK
p38 ATR

P P P P P P P P P P P P

N S8 S9 S15 T18 S20 S33 S37 S45 T55 T81 S149 T150 T155 S215 S313 S314 S315 S366 S376 T377 S378 T387 S392 C
P P P P P P P P P P P

HPK4 TAF1 CK1 Aurora A CHK1 CHK2 CHK1 CHK1

CSNK1A1L GRK5 CHK2 CHK2
ERK2
CSNK1A1L VPK1 CDK5
JNK1 VPK2 CDK7
JNK2 CHK2 CDK9
p38
GSK3ß

Figure 3. p53 phosphorylation sites. Protein kinases and their respective phosphorylation sites on the
p53 protein.

6
Tools for p53 phosphorylation research:

Amino acid Recommended antibody Application

S15 Anti-p53 (phospho S15) antibody IHC-Fr, ICC/IF, IHC-P, WB, IP
S20 Anti-p53 (phospho S20) antibody WB, IP, Dot blot
S33 Anti-p53 (phospho S33) antibody Dot blot, WB, ICC/IF
S37 Anti-p53 (phospho S37) antibody Dot blot, WB
S46 Anti-p53 (phospho S46) antibody WB, IP, IHC-P, ICC/IF

p53 acetylation

p53 is specifically acetylated by p300/CBP and P300/CBP-associated factor (PCAF) in

response to gamma-irradiation and UV light, and TIP60 and hMOF in response to DNA
damage. Acetylation of p53 augments p53 DNA binding, aids in recruiting co-activators,
and stabilizes p53 by inhibiting its ubiquitination by MDM2 (Dai et al., 2010).

Tip60 p300 p300

hMOF CBP CBP Mdm2 p300/CBP Set8

A A A Ub A A A A A M A

N K120 K164 K305 K320 K370 K372 K373 K381 K382 K386 C
A Ub M M Ub Ub Ub Ub Ub

PCAF E4F1 SMYD2 Set7/9 Mdm2

Figure 4. p53 acetylation (A), methylation (M), and ubiquitylation (Ub) sites. Protein kinases and their
respective modification sites on the p53 protein.

Tools to study p53 acetylation:

Amino acid Recommended antibody Application

K370 Anti-p53 (acetyl K370) antibody Flow Cyt, ICC/IF, IP, WB
K373 Anti-p53 (acetyl K373) antibody Flow Cyt, ICC/IF, IHC-P, WB
K381 Anti-p53 (acetyl K381) antibody ELISA, ICC/IF, IHC-P, WB
K382 Anti-p53 (acetyl K382) antibody Flow Cyt, ICC/IF, WB

7
 25 kDa –

Mutant p53 (ab32509)

Etoposide: +1 2-
p53 antibody sampler panel: comprehensive
250 kDa – and specific
150 kDa –
100 kDa –
Detect a comprehensive range of p5375modifications,
kDa – as well as total and mutated
p53 with our comprehensive antibody 50sampler
kDa –
panel.
p53 This panel
(phospho S20) contains
recombinant monoclonal antibodies for precise specificity and exceptionally low
variation between lots. 37 kDa –

T
N 25 kDa –

Antibodies in this panel (ab219089) include the following:

Overview
Mutant p53 (ab32509)
Etoposide: + -

Unmodified p53 mouse monoclonal [DO-1] (ab1101)

Adjacent normal crypts (N) Tumor (T) -- Knockout validated on CRISPR/Cas9-mediated

HAP1 cells
T -- Verified for ChIP, ICC/IF, ELISA, IHC-P, IHC-Fr,
N immunoprecipitation, western blot, flow cytometry
Overview -- Reacts with human p53
-- Cited in over 71 publications

Adjacent normal crypts (N) Tumor (T)

Secondary antibody
only control
Recombinant rabbit monoclonal to Mutant p53 [Y5] (ab32049)

-- Marker of cancer progression

-- Verified use in ICC/IF, western blot, IHC-P, flow
cytometry, immunoprecipitation
-- Tested in human
-- Cited in 29 publications
Secondary antibody
ab32049 MERGED
only control

Secondary antibody
DAPI only control

1 2
250 kDa –
Recombinant
150 kDa – rabbit monoclonal to p53 (phospho S20) [EPR2156(2)]
(ab157454)
100 kDa –
75 kDa –
Phosphorylated p53
ab32049 MERGED
50 kDa – p53 (acetyl K382)
1 2
37 kDa
250 kDa–– -- Verified for use in western blot,
150 kDa –
25 kDa
100 kDa––
immunoprecipitation and dot blot
20 kDa––
75 kDa DAPI
Secondary antibody
only control
-- Tested in human
15 kDa –
10 kDa––
50 kDa p53 (phospho S20)
1 2
250 kDa – p53 (ab179477)
37 kDa –
150 kDa –
GAPDH (ab181602)
100 kDa –
25
75kDa
kDa– –

50 kDa – p53p53
Mutant (acetyl K382)
(ab32509)
Etoposide: + -
37 kDa –

25 kDa –
20 kDa –
15 kDa –
10 kDa –
p53 (ab179477)

T GAPDH (ab181602)

Overview

8
 25 kDa 75
– kDa –

50 kDa – p53(ab32509)
Mutant p53 (phospho S20)
Etoposide: + -

37 kDa –
Recombinant rabbit monoclonal to p53 (phospho S46)
[EP42Y] (ab76242) 25 kDa –

Mutant p53 (ab32509)

Etoposide: + -

T
-- Verified use in western blot, immunoprecipitation,
N IHC-P, ICC/IF
Overview -- Tested in human
-- Cited in 8 publications

T
N
Adjacent normal crypts (N) Tumor (T)
Overview

Recombinant rabbit monoclonal to p53 (phospho S392)

[EP155Y] (ab33889)
Adjacent normal crypts (N) Tumor (T)

-- p53 is phosphorylated on Serine 392 following UV

irradiation (sans gamma irradiation)
Secondary antibody
only control -- Verified use in Dot blot, WB, IHC-P and IP
-- Tested in human, mouse, and rat
-- Cited in 3 publications

Secondary antibody
only control
ab32049 MERGED

Recombinant rabbit monoclonal to p53 (acetyl K382)

[EPR358(2)] (ab75754)
DAPI
Secondary antibody
only control

1 2
250 kDa –
150 kDa – -- Verified use in western blot, ICC/IF and
100 kDa – flow cytometry
75 kDa – ab32049 MERGED -- Tested in human
p53 (acetyl K382)
50 kDa – -- Cited in 18 publications
37 kDa –

25 kDa – Secondary antibody

20 kDa – DAPI only control
15 kDa –
10 kDa –
1 2
250 kDa – p53 (ab179477)

150 kDa –
GAPDH (ab181602)
100 kDa –
75 kDa –

50 kDa – p53 (acetyl K382)

37 kDa –

25 kDa –
20 kDa –
15 kDa –
10 kDa –
p53 (ab179477)

GAPDH (ab181602)

9
References

Bieging, K. T., Mello, S. S., & Attardi, L. D. (2014). Unravelling mechanisms of p53-mediated
tumour suppression. Nature Reviews Cancer, 14(5), 359–370.

Clair, S. S., Giono, L., Varmeh-Ziaie, S., Resnick-Silverman, L., Liu, W. J., Padi, A., …
Manfredi, J. J. (2004). DNA damage-induced downregulation of Cdc25C is mediated
by p53 via two independent mechanisms: One involves direct binding to the cdc25C
promoter. Molecular Cell, 16(5), 725–736.

Dai, C., & Gu, W. (2010). P53 post-translational modification: Deregulated in

tumorigenesis. Trends in Molecular Medicine, 16(11), 528–536.

Inoue, T., Stuart, J., Leno, R., & Maki, C. G. (2002). Nuclear import and export signals
in control of the p53-related protein p73. Journal of Biological Chemistry, 277(17),
15053–15060.

Khoury, M. P., & Bourdon, J. C. (2011). P53 isoforms: An intracellular microprocessor?

Genes and Cancer, 2(4), 453–465.

Martín-Caballero, J., Flores, J. M., García-Palencia, P., & Serrano, M. (2001). Tumor
Susceptibility of p21 Waf1/Cip1-deficient Mice 1. Cancer Research, 61, 6234–6238.

Nag, S., Qin, J., Srivenugopal, K. S., Wang, M., & Zhang, R. (2013). The MDM2-p53
pathway revisited. Journal of Biomedical Research, 27(4), 254–271.

Pascual, M., Mena-Varas, M., Robles, E. F., Garcia-Barchino, M. J., Panizo, C.,
Hervas-Stubbs, S., … Roa, S. (2019). PD-1/PD-L1 immune checkpoint and p53 loss
facilitate tumor progression in activated B-cell diffuse large B-cell lymphomas. In Blood
(Vol. 133).

Perri, F., Pisconti, S., & Vittoria Scarpati, G. Della. (2016). P53 mutations and cancer: A
tight linkage. Annals of Translational Medicine, 4(24), 2–5.

Wade Harper, J., Adami, G. R., Wei, N., Keyomarsi, K., & Elledge, S. J. (1993). The p21
Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell,
75(4), 805–816.

Wang, P., Reed, M., Wang, Y., Mayr, G., Stenger, J. E., Anderson, M. E., … Tegtmeyer, P.
(1994). P53 Domains: Structure, Oligomerization, and Transformation. Molecular and
Cellular Biology, 14(8), 5182–5191.

Williams, A. B., & Schumacher, B. (2016). p53 in the DNA-damage-repair process. Cold
Spring Harbor Perspectives in Medicine, 6(5), 1–16.

10
www.abcam.com
Copyright © 2020 Abcam, All rights reserved

12