p53 protein: guardian of the genome

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

​Contents

• Overview of p53 function and role in cancer 
• p53 protein structure
• MDM2 regulation of p53
• Post-translational modifications of p53
• p53 sampler panel 
• References

Download a pdf version of this guide including additional data

Overview of p53 function and role in cancer

p53 is a tumor suppressor protein and transcription factor 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. 

It activates a multitude of transcriptional targets in response to cellular stress or DNA damage. A broad range of 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). 

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 plays 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).

Download our p53 pathway poster 

p53 protein structure

The p53 protein is active as a tetramer of four 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).

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:

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.​​​

Tools for p53 phosphorylation research:

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).

Tools to study p53 acetylation:

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)

• 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)

• ​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

• Verified for use in western blot, immunoprecipitation, and dot blot
• Tested in human 

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)

• 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)

• ​Verified use in western blot, ICC/IF and flow cytometry
• Tested in human
• Cited in 18 publications