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p53 protein:guardian ofthe genomeReview p53 protein function, structure, and regulation, and find tools and resources to accelerate your p53 research.

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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

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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).

Apoptosis

Gene regulation

Bcl-2

Cyt-c

Bax

Apaf-1/Caspase 9

DNA damage Cellular stress

p53

Mitochondria Cell cycle arrest, cellgrowth inhibition,metabolic stress

inhibition

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

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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) .

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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 CTADI TADII PD DBD OD RD

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

Figure 2. p53 functional domains.

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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

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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.

CDK5PRPKAMPK

ARS ATMATR

ADNA-PKp38

CHK2PLK3

PRAKATR

CDK5DYRK2HPK2PKCδp38

PPPP

JNK CK1 CK1 CHK1CHK2

Aurora ACDK2GSK3ß

GSK3ßPKC

CHK1CHK2PKC

CK2PKR

CDK9p38

ARK5

PPPPPPPP

CSNK1A1LJNK1JNK2

HPK4CSNK1A1L

P P

VPK1VPK2CHK2

CDK5CDK7CDK9p38

GSK3ß

TAF1GRK5ERK2

CK1 Aurora A CHK1CHK2

CHK2 CHK1CHK2

CHK1

P P P P P P P P PN CS8 S9 S15 T18 S20 S33 S37 S45 T55 T81 S149 T150 T155 S215 S313 S314 S315 S366 S376 T377 S378 T387 S392

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

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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).

E4F1PCAF SMYD2 Set7/9 Mdm2

Tip60hMOF

MUbA

N CK120

A A A A A A A

p300CBP

A

A Ub M M Ub Ub Ub Ub Ub

p300CBP Mdm2 Set8p300/CBP

K164 K305 K320 K370 K372 K373 K381 K382 K386

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

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p53 antibody sampler panel: comprehensive and specific

Detect a comprehensive range of p53 modifications, as well as total and mutated p53 with our comprehensive antibody sampler panel. This panel contains recombinant monoclonal antibodies for precise specificity and exceptionally low variation between lots.

Antibodies in this panel (ab219089) include the following:

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

Secondary antibodyonly control

Tumor (T)

Overview

N

T

Adjacent normal crypts (N)

MERGEDab32049

Secondary antibodyonly controlDAPI

250 kDa –

150 kDa –

100 kDa –

75 kDa –

50 kDa –

37 kDa –

25 kDa –20 kDa –15 kDa –10 kDa –

p53 (acetyl K382)

1 2

p53 (ab179477)

GAPDH (ab181602)

250 kDa –150 kDa –

100 kDa –

75 kDa –

50 kDa –

37 kDa –

25 kDa –

p53 (phospho S20)

1 2

Etoposide: + -Mutant p53 (ab32509)

- Knockout validated on CRISPR/Cas9-mediated HAP1 cells

- Verified for ChIP, ICC/IF, ELISA, IHC-P, IHC-Fr, immunoprecipitation, western blot, flow cytometry

- Reacts with human p53 - Cited in over 71 publications

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

Tumor (T)

Overview

N

T

Adjacent normal crypts (N)

MERGEDab32049

Secondary antibodyonly controlDAPI

250 kDa –

150 kDa –

100 kDa –

75 kDa –

50 kDa –

37 kDa –

25 kDa –20 kDa –15 kDa –10 kDa –

p53 (acetyl K382)

1 2

p53 (ab179477)

GAPDH (ab181602)

250 kDa –150 kDa –

100 kDa –

75 kDa –

50 kDa –

37 kDa –

25 kDa –

p53 (phospho S20)

1 2

Etoposide: + -Mutant p53 (ab32509)

- Marker of cancer progression - Verified use in ICC/IF, western blot, IHC-P, flow

cytometry, immunoprecipitation - Tested in human - Cited in 29 publications

Recombinant rabbit monoclonal to p53 (phospho S20) [EPR2156(2)] (ab157454) Phosphorylated p53

Secondary antibodyonly control

Tumor (T)

Overview

N

T

Adjacent normal crypts (N)

MERGEDab32049

Secondary antibodyonly controlDAPI

250 kDa –

150 kDa –

100 kDa –

75 kDa –

50 kDa –

37 kDa –

25 kDa –20 kDa –15 kDa –10 kDa –

p53 (acetyl K382)

1 2

p53 (ab179477)

GAPDH (ab181602)

250 kDa –150 kDa –

100 kDa –

75 kDa –

50 kDa –

37 kDa –

25 kDa –

p53 (phospho S20)

1 2

Etoposide: + -Mutant p53 (ab32509)

- Verified for use in western blot, immunoprecipitation and dot blot

- Tested in human

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Recombinant rabbit monoclonal to p53 (phospho S46) [EP42Y] (ab76242)

- Verified use in western blot, immunoprecipitation, IHC-P, ICC/IF

- Tested in human - Cited in 8 publications

Recombinant rabbit monoclonal to p53 (phospho S392) [EP155Y] (ab33889)

Secondary antibodyonly control

Tumor (T)

Overview

N

T

Adjacent normal crypts (N)

MERGEDab32049

Secondary antibodyonly controlDAPI

250 kDa –

150 kDa –

100 kDa –

75 kDa –

50 kDa –

37 kDa –

25 kDa –20 kDa –15 kDa –10 kDa –

p53 (acetyl K382)

1 2

p53 (ab179477)

GAPDH (ab181602)

250 kDa –150 kDa –

100 kDa –

75 kDa –

50 kDa –

37 kDa –

25 kDa –

p53 (phospho S20)

1 2

Etoposide: + -Mutant p53 (ab32509)

- p53 is phosphorylated on Serine 392 following UV irradiation (sans gamma irradiation)

- Verified use in Dot blot, WB, IHC-P and IP - Tested in human, mouse, and rat - Cited in 3 publications

Recombinant rabbit monoclonal to p53 (acetyl K382) [EPR358(2)] (ab75754)

Secondary antibodyonly control

Tumor (T)

Overview

N

T

Adjacent normal crypts (N)

MERGEDab32049

Secondary antibodyonly controlDAPI

250 kDa –

150 kDa –

100 kDa –

75 kDa –

50 kDa –

37 kDa –

25 kDa –20 kDa –15 kDa –10 kDa –

p53 (acetyl K382)

1 2

p53 (ab179477)

GAPDH (ab181602)

250 kDa –150 kDa –

100 kDa –

75 kDa –

50 kDa –

37 kDa –

25 kDa –

p53 (phospho S20)

1 2

Etoposide: + -Mutant p53 (ab32509)

- Verified use in western blot, ICC/IF and flow cytometry

- Tested in human - Cited in 18 publications

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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.

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