Oligomerization of Mutant p53 R273H is not Required for Gain-of-Function Chromatin Associated Activities

Abstract

The TP53 gene is often mutated in cancer, with missense mutations found in the central DNA binding domain, and less often in the C-terminal oligomerization domain (OD). These types of mutations are found in patients with the rare inherited cancer predisposition disorder called Li-Fraumeni syndrome. We previously found that mutant p53 (mtp53) R273H associates with replicating DNA and promotes the chromatin association of replication-associated proteins mini-chromosome maintenance 2 (MCM2), and poly ADP-ribose polymerase 1(PARP1). Herein, we created dual mutants in order to test if the oligomerization state of mtp53 R273H played a role in chromatin binding oncogenic gain-of-function (GOF) activities. We used site-directed mutagenesis to introduce point mutations in the OD in wild-type p53 (wtp53), and mtp53 R273H expressing plasmids. The glutaraldehyde crosslinking assay revealed that both wtp53 and mtp53 R273H formed predominantly tetramers, while the single OD mutant A347D, and the dual mtp53 R273H-A347D, formed predominantly dimers. The R337C, L344P, mtp53 R273H-R337C, and mtp53 R273H-L344P proteins formed predominantly monomers. Wtp53 was able to activate the cyclin-dependent kinase gene p21/waf and the p53 feedback regulator MDM2. As expected, the transactivation activity was lost for all the single mutants, as well as the mtp53 R273H-dual mutants. Importantly, mtp53 R273H and the dual oligomerization mutants, R273H-A347D, R273H-R337C, and R273H-L344P were able to interact with chromatin. Additionally, the dual oligomerization mutants, R273H-A347D, R273H-R337C, and R273H-L344P, maintained strong interactions with MCM2 and PARP1. Our findings suggest that while mtp53 R273H can form tetramers, tetramer formation is not required for the GOF associated chromatin interactions.

Novel protein contact points among TP53 and minichromosome maintenance complex proteins 2, 3, and 5

“…Their work and ours predict the TP53 interaction with MCM2 to be a weak interaction. 50 A double positive association exists between mutant TP53 R175H and all MCM2-7 complex proteins in both the cytosol and nucleus as determined via proteomic enrichment analysis. 48 Low levels of wild type TP53 were detected interacting with MCM2 in MCF-7 cells 48 and further confirmed with proximity ligation assay.…”

“…Nonetheless, wild type TP53 was found to interact with three MCM proteins which expands recent in vivo results giving evidence of a transient interaction between wild type TP53 and MCM2 and MCM4. 48,50 One explanation for the AVA-Seq method being able to identify these interactions could be the increased sensitivity of the assay. By this, we mean if the binding affinity of wild type TP53 to MCM proteins is higher (weaker or more transient interaction) than the gain-of-function mutation R273H with a given substrate, there would be an increased challenge in detecting interactions.…”

“…Additionally, wild type TP53 interactions are weaker/transient than the GOF mutant TP53 interactions and, therefore more challenging to detect. Work by others demonstrated MCM2 interacted with both wild type TP53 and TP53 R273H 50 . Additionally, they showed TP53 R273H Δ381‐388 (removal of C‐terminal residues) do not seem essential for interaction with MCM2 while deletion of a larger portion (TP53 R273H Δ347‐393) reduced interaction with MCM2 50 .…”

“…46 This method combines bacterial two-hybrid screening with a next-generation DNA sequencing readout of growth changes of the host cells signifying potential protein-protein interactions. Our previous study 46 and the work of others [47][48][49][50][51] indicated interactions among MCM complex proteins and TP53 (also see Table S5). The interactions between MCM2|MCM5, 1,19,52-54 MCM2|MCM3 [52][53][54][55] and MCM5|MCM3 1,19,[52][53][54][55][56] are well documented.…”

ALK-Positive Anaplastic Large-Cell Lymphoma in a Patient with Chronic Lymphocytic Leukemia: A Case Report and Literature Review

“…This mutation was proposed to promote oncogenic gain-of-function activity by destabilizing p53 tetramer formation. 16 R273H mutation can also drive AKT signaling and suppress BMF expression, resulting in enhanced cell survivability and anoikis resistance. 17 Additionally, R273H mutation can promote colorectal cancer stem cells, which leads to enhanced cancer self-renewal, tumor propagation and chemoresisitance.…”