Review p53 protein function, structure, and regulation ...

1 1p53 protein :guardian ofthe genomeReview p53 protein function, structure, and regulation , and find tools and resources to accelerate your p53 of p53 function and role in cancer ..2 -Cell cycle arrest ..3 -DNA repair ..3 -p53 gene mutation and cancer ..3p53 protein structure ..4 MDM2 regulation of p53 ..5 Post-translational modifications of p53 ..6 -p53 phosphorylation ..6 -p53 acetylation ..7p53 antibody sampler panel ..8 References ..102 Overview of p53 function and role in cancerp53 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 we look at the function, structure, and modifications of p53 and outline the tools you can use to accelerate your p53 is a transcription factor that activates a multitude of transcriptional targets in response to cellular stress or DNA damage.

2 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). ApoptosisGene regulationBcl-2 Cyt-cBaxApaf-1/Caspase 9 DNA damageCellular stressp53 MitochondriaCell cycle arrest, cellgrowth inhibition,metabolic stressinhibitionFigure 1. Mechanisms of p53 function in apoptosis.. p53 responds to DNA damage or cellular stress and activates the Bax and Apaf-1 cycle arrestp53 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.)

3 , 1993). p53 can also bring about cell cycle arrest at the G2/M phase through binding to other p53 target genes such as 14-3-3 (Mart n-Caballero et al., 2001) and cdc25C (Clair et al., 2004). DNA repairp53 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.

4 , 2016) . p53 gene mutation and cancerTP53 (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) .4p53 protein structureThe 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.

5 , 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 C-terminal NES, a highly conserved region has been shown to be essential for nuclear export of p53.

6 Both the NLS and NES regions are required for nuclear-cytosolic shuttling of p53 to regulate p53 transcriptional function (Inoue et al., 2002).NCTADITADIIPDDBDODRD1-4243-6263-97 102-292323-356363-393 Figure 2. p53 functional 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 AntibodyAnti-MDM2 [2A10] antibodyAnti-MDM2 (phospho S166) antibody6 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.)

7 , 2010). Here we focus on some of these post-translational modifications (phosphorylation and acetylation) and their role in p53 stress response. p53 phosphorylationPhosphorylation 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 ATMATRADNA-PKp38 CHK2 PLK3 PRAKATRCDK5 DYRK2 HPK2 PKC p38 PPPPJNKCK1CK1 CHK1 CHK2 Aurora ACDK2 GSK3 GSK3 PKCCHK1 CHK2 PKCCK2 PKRCDK9p38 ARK5 PPPPPPPPCSNK1A1 LJNK1 JNK2 HPK4 CSNK1A1 LPPVPK1 VPK2 CHK2 CDK5 CDK7 CDK9p38 GSK3 TAF1 GRK5 ERK2CK1 Aurora ACHK1 CHK2 CHK2 CHK1 CHK2 CHK1 PPPPPPPPPNCS8 S9 S15 T18S20S33S37S45T55T81S149T150T155S215S 313S 314S 315S366S376T377S378T387S392 Figure 3.

8 P53 phosphorylation sites. protein kinases and their respective phosphorylation sites on the p53 protein . 7 Tools for p53 phosphorylation research: Amino acidRecommended antibodyApplicationS15 Anti-p53 (phospho S15) antibodyIHC-Fr, ICC/IF, IHC-P, WB, IPS20 Anti-p53 (phospho S20) antibodyWB, IP, Dot blotS33 Anti-p53 (phospho S33) antibodyDot blot, WB, ICC/IFS37 Anti-p53 (phospho S37) antibodyDot blot, WBS46 Anti-p53 (phospho S46) antibodyWB, IP, IHC-P, ICC/IFp53 acetylationp53 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.)

9 , 2010).E4F1 PCAFSMYD2 Set7/9 Mdm2 Tip60hMOFMUbANCK120 AAAAAAAp300 CBPAAUbMMUbUbUbUbUbp300 CBPMdm2 Set8p300/CBPK164K305K320K370K372K373K381 K382K386 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 acidRecommended antibodyApplicationK370 Anti-p53 (acetyl K370) antibodyFlow Cyt, ICC/IF, IP, WBK373 Anti-p53 (acetyl K373) antibodyFlow Cyt, ICC/IF, IHC-P, WBK381 Anti-p53 (acetyl K381) antibodyELISA, ICC/IF, IHC-P, WBK382 Anti-p53 (acetyl K382) antibodyFlow Cyt, ICC/IF, WB8p53 antibody sampler panel: comprehensive and specificDetect a comprehensive range of p53 modifications, as well as total and mutated p53 with our comprehensive antibody sampler panel.

10 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 controlTumor (T)OverviewNTAdjacent normal crypts (N)MERGEDab32049 Secondary antibodyonly controlDAPI250 kDa 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa 20 kDa 15 kDa 10 kDa p53 (acetyl K382)1 2p53 (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 publicationsRecombinant rabbit monoclonal to Mutant p53 [Y5] (ab32049)Secondary antibodyonly controlTumor (T)OverviewNTAdjacent normal crypts (N)MERGEDab32049 Secondary antibodyonly controlDAPI250 kDa 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa 20 kDa 15 kDa 10 kDa p53 (acetyl K382)1 2p53 (ab179477)GAPDH (ab181602)250 kDa 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa p53 (phospho S20)1 2 Etoposide.